Magnetic biological entity separation device and method of use

ABSTRACT

The current invention relates to the method and apparatus to magnetically separate biological entities with magnetic labels from a fluid sample. The claimed methods separate biological entities with magnetic labels by using a magnetic device. The claimed methods further include processes to dissociate biological entities magnetic conglomerate after magnetic separation.

BACKGROUND

The current invention generally relates to methods and apparatus toseparate biological entities, including cells, bacteria and moleculesfrom human blood, body tissue, body fluid and other human relatedbiological samples. The disclosed methods and apparatus may also beutilized to separate biological entities from animal and plant samples.More particularly, the current invention relates to the methods andapparatus for achieving separation of biological entities with using oneor more of a micro-fluidic separation device/chip (“UFL”), and one ormore of a magnetic separation device (“MAG”), individually or incombination. For description purpose, “cells” will be used predominantlyhereafter as a typical representation of biological entities in general.However, it is understood that the methods and apparatus as disclosed inthis invention may be readily applied to other biological entitieswithout limitation.

Separation of biological entities from a fluid base solution, forexample separating a specific type of white blood cells from humanblood, typically involves a first step of identifying the targetbiological entities with specificity, and followed by a second step ofphysical extraction of the identified target biological entities fromthe fluid base solution. In human blood, different types of biologicalcells may have various types of surface antigens or surface receptors,which are also referred to as surface markers in this invention. Certainsurface markers on a given type of cells may be unique to said type ofcells and may be used to identify said type of cells from blood samplewith specificity.

FIG. 1A through FIG. 1C show examples of identifying or labeling targetcells 1, with using superparamagnetic labels 2 (“SPL”) as in FIG. 1A,using optical fluorescent labels 3 (“OFL”) as in FIG. 1B, and using boththe SPL 2 and OFL 3 together as in FIG. 1C.

In FIG. 1A, cell 1 has surface markers 11. SPLs 2 are conjugated withsurface antibodies or ligands, also referred to as “probe” 21, whichspecifically bind to the surface markers 11 of cell 1. Large quantity ofSPL 2 having probes 21 are put into the solution where cell 1 resides.After incubation processes 9, a plurality of SPLs 2 are bound to cell 1surface with probes 21 selectively bound to surface markers 11 withspecificity. Thus, cells 1 is magnetically identified or labeled by SPLs2, i.e. magnetically labeled cell 10. A magnetic field with sufficientfield gradient may be applied to cell 10 to produce a physical force onthe SPLs 2 attached to the cells 10 surface. With sufficient strength,the physical force working through the SPLs 2 on cell 10 may be used toseparate and physically remove cell 10 from its liquid solution.

In FIG. 1B, cell 1 has surface markers 12. OFLs 3 are conjugated withprobes 22, which specifically bind to the surface markers 12 of cell 1.Large quantity of OFLs 3 having probes 23 are put into the solutionwhere cell 1 resides. After incubation processes 9, a plurality of OFLs3 are bound to cell 1 surface with probes 22 selectively bound tosurface markers 12 with specificity. Thus, cells 1 is opticallyidentified or labeled by OFLs 3, i.e. optically labeled cell 20. Byusing an optical based cell separation system, cell 1 may be separatedfrom its liquid solution based on the optical signal that OFL 3 producesunder an excitation light. One type of such optical based cellseparation system is a flow cytometer, wherein said liquid solution isstreamed through a conduit within said flow cytometer as a continuousflow. At least one excitation light source produces a light spot uponsaid liquid flow through said conduit at a first optical wavelength. Inpresence of OFL 3 in the light spot, OFL 3 is excited by firstwavelength and radiates optical light at a second wavelength. When cell1 with bound OFLs 3 passes said light spot within said flow, OFLs 3bound to cell 1 radiate optical signal in second wavelength, whereasstrength of said optical signal as well as duration while cell 1 passesthe light spot may be used to identify presence of cell 1 by the flowcytometer, which then diverts cell 1 into a second liquid flow path ormechanically remove cell 1 from the liquid flow, thus separating cell 1from fluid base. In practice, OFL 3 bound to cell 1 may be in varioustypes of fluorescent dyes or quantum dots, producing exited opticallight at multiple wavelengths. A plurality of excitation light sourcesmay also be used in same flow cytometer system to produce excitationlight spots at different locations of the liquid flow with differentexcitation light wavelength. Combination of various wavelength producedby OFL 3 on same cell 1 may be used to increase specificity ofseparation of cell 1, especially when a combination of various types ofsurface markers 12 is needed to specifically identify a sub-categorytarget cell 1 population from a major category of same type of cells,for example CD4-T cells from other white blood cells.

In FIG. 1C, cell 1 has both surface markers 11 and 12. SPLs 2 conjugatedwith probes 21 and OFLs 3 conjugated with probes 22 are both bound tocell 1 surface after incubation processes 9 to form magnetically andoptically labeled cell 30. Cell 30 allows for separation of cell 30 witha combination of magnetic separation and an optical based cellseparation system, where a the magnetic separation through SPLs 2 mayprovide a fast first stage separation of cell category including cell30, while the optical separation through OFLs 3 may provide a secondstage separation of cell 30 after magnetic separation with morespecificity. Alternatively, cell 30 may be separated via OFLs 3 in afirst stage and via SPLs 2 in a second stage. In either case, SPLs 2 andOFLs 3 together may help increase speed, efficiency and specificity inseparation of cell 1 compared with FIG. 1A and FIG. 1B.

FIG. 2A shows an example of conventional magnetic separation through SPL2. In a container 5, liquid solution 6 contains cells 10 of FIG. 1A orcells 30 of FIG. 1C that are bound with plurality of SPLs 2 on cellsurface. Magnet 4, preferably a permanent magnet, is positioned inproximity to wall of container 5. Magnet 4 has a magnetizationrepresented by arrow 41 indicating a north pole (“N”) and a south pole(“S”) on top and bottom surfaces of the magnet 4. Magnetic fieldproduced by the magnetization 41 in the solution 6 is higher at thecontainer 5 wall directly opposing the N surface of the magnet 4, andlower at locations within solution 6 further away from the magnet 4,thus creating a magnetic field gradient pointing towards the magnet 4within the solution 6. SPLs 2 bound to cell 10/30 are superparamagnetic,which are effectively non-magnetic in absence of magnetic field, butwill gain magnetic moment in presence of the magnetic field produced bythe magnet 4. With the magnetic moment of SPLs 2 and the magnetic fieldgradient from magnet 4, cells 10/30 will be pulled by the force producedby the magnetic field from magnet 4 towards magnet 4. After sufficienttime 7, cells 10/30 may be depleted from solution 6 and formconglomerate at inside surface of the container 5 wall opposing magnet4. In conventional practice, solution 6 may be removed from container 5,while maintaining magnet 4 position relative to container 5 thus cells10/30 are retained as conglomerate against container 5 inside surface.Afterwards, magnet 4 may be removed from container 5. With absence ofmagnetic field, conglomerate of cells 10/30, together with any non-boundfree SPLs in the conglomerate, shall self-demagnetize over extensivetime to be non-magnetic and cells 10/30 may be removed from container 5as individual cells 10/30.

Conventional method as shown in FIG. 2A has limitations in actualapplications. For the SPL 2 to be superparamagnetic, the size of thefundamental superparamagnetic particles (“SPN”) contained in SPL 2, forexample iron oxide particles, shall be in the range of 10 nm (nanometer)to 30 nm, where a smaller particle size makes the particles moreeffectively superparamagnetic but harder to gain magnetic moment inpresence of magnetic field, while a larger particle size makes theparticles more difficult to become non-magnetic when magnetic field isremoved. SPL 2 is typically composed of SPNs dispersed in a non-magneticmatrix. For example, certain SPL 2 is a solid sphere formed by SPNsevenly mixed within a polymer base, typically in the size of larger than1 um (um). In another case, SPL 2 is solid bead formed by SPNs mixedwithin an oxide or nitride base, for example iron oxide nanoparticlesmixed in silicon oxide base, which can be in the size of a few hundrednanometers or tens of nanometers. For the cells 10/30 of FIG. 2A to besuitable for additional cellular processes, including cell culture andcell analysis, SPL 2 size is desirable to be smaller than the cellitself, which is usually a few ums. Thus, SPL 2 with sub-um size (<1 um)is desired. SPL 2 size less than 500 nm is more preferred. SPL 2 sizeless than 200 nm is most preferred. However, when SPL 2 average size issmaller, variation of SPL 2 size becomes larger statistically. FIG. 2Bshows example schematics of single SPL 2 magnetic moment in the presenceof an applied magnetic field. Solid curve 22 indicates SPL 2 having apopulation nominal size, or average size, where SPL 2 magnetic momentincreases with higher magnetic field. With magnetic field strengthincreasing from 0 to Hs, nominal size SPL magnetic moment increases withfield strength in a linear trend at beginning, until reaching asaturation region where magnetic moment plateaus to Ms, which isdetermined by the saturation moment of the SPNs material within the SPL2. For SPL 2 with a smaller size than nominal size, curve 23 indicatesthat at the same magnetic field strength, smaller size SPL 2 gains alower moment, and thus experiencing a lower magnetic force, and requiresa higher field to reach saturation magnetic moment Ms. For a larger sizeSPL 2 than nominal size, curve 24 indicates larger size SPL 2 is easierto saturate to Ms with a lower field and gains a higher moment at samemagnetic field strength.

Now referring back to FIG. 2A, for SPL 2 with sub-um size that issuitable for cell separation and cellular processes, conventional methodof FIG. 2A has limitation of not being able to produce high magneticfield strength and strong magnetic field gradient in solution 6 atlocations further away from the container 5 wall opposing magnet 4 Nsurface. Therefore, smaller size SPL 2 of curve 23 of FIG. 2B at fartherend of the container 5 from magnet 4 may be difficult to magnetize bymagnet 4 field and experiences smaller force to move the cell 10/30towards magnet 4. To reach complete depletion of cells 10/30 in solution6 within container 5, it may require significant amount of time.Meanwhile, volume of container 5 is limited also due to magnetic fieldstrength from magnet 4 may not be sufficient to magnetize the smallerSPL 2 of curve 23 of FIG. 2B at large container 5 sizes. Besides overallprocess being slow, another drawback in conventional method of FIG. 2Ais that the operation as described in FIG. 2A typically involves airexposure of cells 10/30 conglomerate during the steps of solutionremoval and later removal of cells 10/30 from container 5. Such airexposure poses challenge in achieving sterile separation of cells 10/30for clinical purpose, as well as risk of cell 10/30 damage or death thatnegatively affects further cellular processes of cell 10/30.

FIG. 3A shows another example of magnetic separation of cells 10/30 withSPL 2 in prior art. In FIG. 3A, solution 6 containing cells 10/30 ispassed through a column 31 that is filled with ferromagnetic orferromagnetic spheres 36. By applying a magnetic field across the columnwith magnets 32 and 33, where dashed lines 34 indicates applied magneticfield direction, spheres 36 may be magnetized by the field and producinglocalized magnetic field in gaps between neighboring spheres 36. Suchlocal field and field gradient between spheres 36 gaps may be strong,due to the small dimensions of the gaps, to effectively magnetize SPL 2of all sizes when SPL 2 in solution 6 passes through the gaps betweenthe spheres 35 during a downward flow of solution 6 as indicated byarrow 35, where SPL 2 may be attracted to various spheres 36 surface andseparated from the solution 6. Prior art of FIG. 3A may effectivelyavoid the air exposure issue of FIG. 2A, and may possess at a higherseparation speed of cells 10/30 than FIG. 2A during the flow 35.However, an intrinsic issue of FIG. 3A method is that with the spheres36 being ferromagnetic or ferromagnetic and is much larger in size thancells 10/30, magnetic domains in spheres 36 will exist even afterremoval of magnets 32 and 33 from the column 31. Such magnetic domains,and domain walls between the domains, will inevitably produce localmagnetic field around the surface of the spheres 36, which will keep theSPLs 2 on cells 10/30 magnetized and strongly attracting the cells 10/30when magnets 32 and 33 are removed. Therefore, the cells 10/30 areinherently more difficult to be removed from the column 31 in FIG. 3Athan FIG. 2A. Cells 10/30 loss due to not completely removed from column31 after separation is inherently high. In certain prior art method, apressurized high speed buffer flow may be used to force wash the cells10/30 from the spheres in column 36. However, such forced flow willinevitably causes mechanical damage to the cells and will still leavesignificant percentage of cells 10/30 in column 31 due to the strongdomain wall field of spheres 36. Besides cells 10/30 loss, anotherintrinsic issue of FIG. 3A method is introducing spheres 36 as foreignmaterials in the flow of solution 6, which is not desirable for sterileprocess needed for clinical applications.

FIG. 3B then shows another prior similar to method of FIG. 3A, exceptmesh 37 made of ferromagnetic or ferromagnetic wires are introduced inthe column 31 instead of spheres or blocks 36. When magnetic field 34 isapplied by the magnets 32 and 33, wires of mesh 37 are magnetized andadjacent wires of mesh 37 produce local magnetic field around the wires.Clearances between wires of the mesh allow fluid 6 to flow in direction35 within the column. When cells 10/30 is in proximity to wires of mesh37, cells 10/30 may be attracted to the wire surface due to the localmagnetic field and field gradient produced by the wires of the mesh 37.Compared to FIG. 3A prior art, FIG. 3B may adjust size of wires and sizeof clearance of mesh 37 to tradeoff between cells 10/30 separation speedand cell loss in column. However, in practice, due to the gap betweenspheres 36 is much smaller than clearance size in mesh 37, cells 10/30separation speed in FIG. 3B is slower than FIG. 3A, while FIG. 3B stillpossesses the same cells loss issue of FIG. 3A, where domains in thewires of mesh 37 maintains SPL 2 magnetic moment after magnets 32 and 33are removed and cells 10/30 are attracted to the wires by the domain anddomain wall. Cells 10/30 loss due to the magnetic domains in wires ofmesh 37 also exists in FIG. 3B. Additionally, FIG. 3B is same as FIG. 3Ain introducing mesh 37 as foreign materials in the flow of solution 6,which is not desirable for sterile process.

FIG. 3C shows another prior art, where magnets 32 and 33 are eachattached with a soft magnetic flux guide 38 with an apex. The fluxguides 38 produce localized magnetic field between the apexes of theguides 38 with high field strength and high gradient close to theapexes. FIG. 3C shows the cross-sectional view of the conduit 39, whichis intrinsically a circular tubing, whereas solution 6 containing cells10/30 flows along the tubing 39 length in the direction perpendicular tothe cross-section view. Tubing 39 is positioned on one side of the gapof the apexes. Magnetic field lines 34 exhibit a higher density closerto the gap indicates both higher magnetic field strength and highermagnetic field gradient towards the gap. Magnetic field 34 produceseffective force on cells 10/30 in solution 6 and pulls the cells 10/30from solution 6 towards the tubing 39 inside wall that is closest to theapexes of the guides 38. Prior art of FIG. 3C when compared to prior artof FIG. 3A and FIG. 3B has the advantages of: (1) not introducingforeign material in the flow path; (2) when magnets 32 and 33 areremoved from tubing together with guides 38, there is no ferromagneticor ferromagnetic sphere 36 or mesh 37 in the tubing, thus avoiding thedomain structures related loss of cells 10/30.

However, prior art of FIG. 3C also has intrinsic deficiencies. Firstdeficiency is the flow speed of solution 6, or flow rate, in the tubing39 is limited by the prior art design of FIG. 3C. The separation speedof cells 10/30 of prior art as in FIG. 3C is not sufficient for manyapplications. Circular tubing conduit 39 as shown in FIG. 3C experienceshigh field and high field gradient at lower end of tubing 39, wherecells 10/30 closer to the lower end of tubing 39 may experience a highforce that pulls them to move towards the tubing 39 lower wall innersurface much faster. However, for the cells 10/30 closer to the top endof the tubing 39, due to the narrow wedge gap and position of the tubing39 being on one side of the gap, magnetic field and gradient issignificantly lower than the lower end. Thus cells 10/30 closer to thetop end of the tubing 39 experiences a much smaller force and moves tolower end of tubing 39 at a much slower speed. For a limited length ofthe tubing 39 in the perpendicular to cross-sectional view direction,all cells 10/30 within the fluid 6 flowing through the tubing 39 need tobe separated from solution 6 to form a conglomerate on the insidesurface of the tubing close to the apexes before solution 6 exits thetubing 39. Due to slower speed of cells 10/30 moving from top of thetubing 39, flow rate of solution 6 needs to be slow such that it canallow enough time for all the cells 10/30 near top of tubing 39 to beattracted into the conglomerate. If solution 6 flows through the tubing39 at higher speed, it will cause incomplete separation of cells 10/30from solution. Such limitation on flow rate due to the circular designof tubing 39, where tubing top end being further away from high fieldand high gradient apexes cannot be cured by a smaller size tubing 39. Asmaller cross-sectional size circular tubing 39 will bring the top endof the tubing 39 closer to the wedge gap. However, due to the smallercross-section size, volume of solution 6 flowing through the tubing 39in a unit time frame, i.e. flow rate of solution 6, will reduce whenflow speed of solution 6 maintains. To maintain same flow rate as in alarger tubing 39, solution 6 flow speed needs to increase, which thengives less time for cells 10/30 at top end of smaller size tubing 39 tomove to the conglomerate site, and offsets the effect of small sizetubing 39.

A second deficiency of FIG. 3C prior art is the inability to dissociateindividual cell 10/30 from conglomerate of cells 10/30 and non-boundfree SPL 2, as the conglomerate will not self-demagnetize with easeafter magnets 32 and 33, together will guides 38, are removed fromtubing 39 in actual applications. Demagnetization of SPL 2 relies on theSPNs within SPL 2 being effectively nanoparticles. However, as theconglomerate forms an effective larger body of superparamagneticmaterial, the SPNs within SPL 2 experiences magneto-static field from alarge number of closely packed SPNs from neighboring SPL 2 in theconglomerate, which reduces the super-paramagnetic nature of the SPNs.In one case, the SPL 2 of cells 10/30 within conglomerate requiresextensive time to self-demagnetize, which is not practical for manyapplications. In another case, the conglomerate won't self-demagnetizedue to the SPN being more ferromagnetic in conglomerate form, which isundesirable. High pressure flushing as utilized in FIG. 3A is noteffective in FIG. 3C, as majority of the circular tubing 39 inner areais occupied by empty space, while conglomerate is compacted on the lowerend of tubing 39, such flush will mainly flow through the top section ofthe tubing 39 without producing enough friction force on theconglomerate of cells 10/30 to remove the cells 10/30 from the tubing 39lower wall. As prior art does not provide an effective method todissociate conglomerate and remove cells 10/30 from tubing 39, suchdeficiency of FIG. 3C prior art is limiting its application.

Prior arts are limited either in causing cell loss and introducingforeign materials in the flow path, or limited in the flow rate ofsolution 6 and the ability to extract separated cells from conglomeratewith an effective dissociation method.

It is desired to have a method and an apparatus that can achieve highflow rate magnetic separation of cells 10/30 without introducing foreignmaterial in the flow path of the biological solution, and being able todissociate cells 10/30 from conglomerate in a practically short timewithout damaging the cells.

SUMMARY OF THE INVENTION

This invention describes novel methods and novel magnetic separationdevices (“MAG”) that are able to: (1) separate biological entities boundwith SPL from biological solution with high flow rate, without exposureof biological entities to air, and without introducing foreign materialin the flow path of the biological solution carrying the biologicalentities; (2) dissociate the biological entities from the magneticallyseparated conglomerate.

This invention further describes novel methods and novel micro-fluidicseparation devices (“UFL”) that separate biological entities frombiological solutions based on the size of the biological entities.

This invention further describes novel methods and novel apparatus usingMAG and UFL individually and in combination to separate biologicalentities from biological solutions for various separation applications.

This invention further describes methods of pre-symptom early tumordetection and methods of using MAG and UFL during the process of tumordetection.

The methods, components and apparatus as disclosed by this invention maybe utilized to separate biological entities, including cells, bacteriaand molecules, from human blood, human body tissue, human bones, humanbody fluid, human hairs, other human related biological samples, as wellas biological entities from animal and plant samples alike withoutlimitation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates superparamagnetic labels (SPL) binding to a cell.

FIG. 1B illustrates optical fluorescent labels (OFL) binding to a cell.

FIG. 1C illustrates SPLs and OFLs binding to a cell.

FIG. 2A illustrates cells bound with SPLs being separated by a magnet.

FIG. 2B is a plot of SPL magnetization vs magnetic field strength fordifferent SPL sizes.

FIG. 3A is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 3B is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 3C is a cross-sectional view of a prior art magnetic cellseparator.

FIG. 4 is a cross-sectional view of the first embodiment of a magneticseparation device (“MAG”) with a “C” shape rigid channel.

FIG. 5 is a cross-sectional view of the first embodiment of MAG having a“C” shape rigid channel in separation position.

FIG. 6 is a cross-sectional view of the first embodiment of MAG having a“C” shape rigid channel in separation position with cells beingseparated.

FIG. 7 is a side view of FIG. 6.

FIG. 8A is a cross-sectional view of the second embodiment of MAG.

FIG. 8B is a cross-sectional view of the third embodiment of MAG.

FIG. 9 a cross-sectional view of the first embodiment of MAG with aflexible channel.

FIG. 10 illustrates the first embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 11 illustrates the first embodiment of MAG having a flexiblechannel in lifted position after cell separation.

FIG. 12 illustrates cross-sectional view of the fourth embodiment of MAGhaving a “D” shape rigid channel in separation position.

FIG. 13 illustrates cross-sectional view of the fourth embodiment of MAGwith a flexible channel.

FIG. 14 illustrates cross-sectional view of the fourth embodiment of MAGhaving a flexible channel in separation position with cells beingseparated.

FIG. 15A illustrates cross-sectional view of the fifth embodiment ofMAG.

FIG. 15B illustrates cross-sectional view of the sixth embodiment ofMAG.

FIG. 15C illustrates cross-sectional view of the seventh embodiment ofMAG.

FIG. 16 illustrates cross-sectional view of a pair of third embodimentof MAGs with a pair of flexible channels on a single channel holder.

FIG. 17 illustrates cross-sectional view of a pair of third embodimentof MAGs with a pair of flexible channels on a single channel holder inseparation position.

FIG. 18 illustrates cross-sectional view of four of fifth embodiment ofMAGs with four flexible channels on a single channel holder.

FIG. 19 illustrates cross-sectional view of four of fifth embodiment ofMAGs with four flexible channels on a single channel holder inseparation position.

FIG. 20A illustrates cross-sectional view of the eighth embodiment ofMAG with a rotated “D” shape rigid channel in separation position.

FIG. 20B illustrates cross-sectional view of the eighth embodiment ofMAG with a flexible channel.

FIG. 20C illustrates cross-sectional view of the eighth embodiment ofMAG having a flexible channel in separation position with cells beingseparated.

FIG. 21A illustrates cross-sectional view of the ninth embodiment of MAGhaving a “V” shape rigid channel in separation position.

FIG. 21B illustrates cross-sectional view of the ninth embodiment of MAGwith a flexible channel.

FIG. 21C illustrates cross-sectional view of the ninth embodiment of MAGhaving a flexible channel in separation position with cells beingseparated.

FIG. 22A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated, and ademagnetization (“DMAG”) magnet positioned over and away from MAG.

FIG. 22B illustrates the flexible channel of FIG. 22A departing MAG andmoving into position where flexible channel holder is in close proximityto, or contacts, the DMAG magnet.

FIG. 22C illustrates the cells in the flexible channel of FIG. 22B beingdissociated from conglomerate by the DMAG magnet.

FIG. 22D illustrates the flexible channel of FIG. 22C moving into a lowmagnetic field position between MAG and DMAG magnet.

FIG. 23A illustrates mechanical vibration is applied to a flexiblechannel holder by a motor after cells are magnetically separated insidethe flexible channel.

FIG. 23B illustrates ultrasound vibration is applied to a flexiblechannel holder by a piezoelectric transducer (“PZT”) after cells aremagnetically separated inside the flexible channel.

FIG. 23C illustrates mechanical vibration is applied to a flexiblechannel by a motor after cells are magnetically separated inside theflexible channel.

FIG. 23D illustrates ultrasound vibration is applied to a flexiblechannel by a PZT after cells are magnetically separated inside theflexible channel.

FIG. 23E is a side view of the flexible channel of FIG. 22D.

FIG. 24A illustrates the third embodiment of MAG having a flexiblechannel holder in close proximity to, or in contact with, a DMAG magnetafter cells are magnetically separated by MAG, where DMAG magnet ispositioned on the side and away from MAG.

FIG. 24B illustrates the flexible channel of FIG. 24A rotating into alow magnetic field position between MAG and DMAG magnet.

FIG. 25A illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet.

FIG. 25B illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet attached with a softmagnetic pole.

FIG. 25C illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is a permanent magnet attached with a pairof soft magnetic poles.

FIG. 25D illustrates a flexible channel holder at demagnetizationposition, where DMAG magnet is an electro-magnet.

FIG. 25E illustrates flexible channel holder at demagnetizationposition, where mechanical vibration is applied to DMAG magnet by amotor.

FIG. 25F illustrates flexible channel holder at demagnetizationposition, where ultrasound vibration is applied to DMAG magnet by a PZT.

FIG. 26A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 26B illustrates the flexible channel of FIG. 26A departs from MAGand rotates.

FIG. 26C illustrates the conglomerate of separated cells in the flexiblechannel of FIG. 26B being rotated to top end of the flexible channel.

FIG. 26D illustrates the flexible channel of FIG. 26C moves todemagnetization position.

FIG. 27A illustrates the third embodiment of MAG having a flexiblechannel in separation position with cells being separated.

FIG. 27B illustrates the flexible channel and its holder of FIG. 27Adepart from MAG.

FIG. 27C illustrates mechanical vibration is applied to the channelholder by a motor.

FIG. 27D illustrates ultrasound vibration is applied to the channelholder by a PZT.

FIG. 28A illustrates a side view of a flexible channel where thereflexible channel is mechanically stretched.

FIG. 28B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 28A.

FIG. 29A illustrates a side view of a flexible channel where theflexible channel is mechanically compressed.

FIG. 29B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 29A.

FIG. 30A illustrates a side view of a flexible channel where theflexible channel is mechanically twisted.

FIG. 30B illustrates cells being dissociated from conglomerate afterremoval of the external force of FIG. 30A.

FIG. 31 is a schematic diagram illustrating methods to use MAG tomagnetically separate biological entities from fluid solution.

FIG. 32 illustrates a method to align a flexible channel MAG wedge of aMAG device.

FIG. 33A illustrates a flexible channel attached to the output port of aperistaltic pump, where a flow limiter is attached to the flexiblechannel to reduce the flow rate pulsation.

FIG. 33B illustrates a top-down view of the inner structure of a firsttype flow limiter.

FIG. 33C illustrates a side view of a second type flow limiter.

FIG. 34A illustrates FIG. 33A flow limiter being disengaged from theflexible channel.

FIG. 34B is a schematic illustration of fluid flow rate with largepulsation.

FIG. 35A illustrates FIG. 33A flow limiter being engaged upon theflexible channel.

FIG. 35B is a schematic illustration of fluid flow rate with reducedpulsation.

FIG. 36A illustrates FIG. 33A flow limiter causing pressure built up atthe fluid incoming end of the flow limiter.

FIG. 36B illustrates FIG. 36A flow limiter being disengaged and causinghigh speed fluid pulse that pushes the dissociated cells of FIG. 36A outof the channel.

FIG. 37 is a schematic illustration of fluid flow rate pulse created bythe process of FIG. 36A to FIG. 36B transition where flow limiter isdisengaged.

FIG. 38A is a top-down view of a micro-fluidic chip (“UFL”).

FIG. 38B is a cross-sectional view of a portion of the FIG. 38A UFLincluding entity fluid inlet, buffer fluid inlet, and part of the UFL.

FIG. 38C is a schematic diagram illustrating a single fluidic pressurenode created between two side walls of the UFL of FIG. 38A by ultrasoundvibration generated by a PZT.

FIG. 38D is a schematic diagram illustrating the fluid acoustic wave ofFIG. 38C causing larger size entities to move around center of the UFL.

FIG. 39 is a schematic diagram illustrating methods to use UFL toseparate biological entities of different sizes.

FIG. 40A is a cross-sectional view of a portion of first embodiment UFLwhich includes a uniformly formed soft magnetic layer.

FIG. 40B is a schematic diagram illustrating the fluid acoustic wave andentities separation in UFL of FIG. 40A in presence of a magnetic field.

FIG. 40C is a schematic diagram illustrating a protection layerconformably deposited around the UFL surface before attached cap.

FIG. 41A is a top-down view of a second embodiment UFL including a widechannel and a narrow channel in sequential arrangement.

FIG. 41B is a cross-sectional view of second embodiment UFL across widechannel.

FIG. 41C is a cross-sectional view of second embodiment UFL acrossnarrow channel.

FIG. 42A is a top-down view of a third embodiment UFL including a widechannel and a narrow channel, and side channels from wide channel tonarrow channel transition section.

FIG. 42B is a cross-sectional view of third embodiment UFL across widechannel.

FIG. 42C is a cross-sectional view of third embodiment UFL across narrowchannel and side channels.

FIG. 43 is a top-down view of a fourth embodiment UFL having three-stagechannel width reduction along channel flow direction, and side channelsfrom transition sections.

FIG. 44A illustrates first type sample processing method including UFLand MAG, with a first type flow connector connecting the UFL largeentity outlet and MAG inlet.

FIG. 44B illustrates first type sample processing method with a secondtype flow connector connecting the UFL large entity outlet and MAGinlet.

FIG. 44C illustrates first type sample processing method with a thirdtype flow connector connecting the UFL large entity outlet and MAGinlet.

FIG. 45A illustrates second type sample processing method including UFLand MAG, with a first type flow connector connecting the UFL smallentity outlet and MAG inlet.

FIG. 45B illustrates second type sample processing method with a secondtype flow connector connecting the UFL small entity outlet and MAGinlet.

FIG. 45C illustrates second type sample processing method with a thirdtype flow connector connecting the UFL small entity outlet and MAGinlet.

FIG. 46A illustrates third type sample processing method including MAGan dUFL, with a first type flow connector connecting the MAG outlet andUFL entity fluid inlet.

FIG. 46B illustrates third type sample processing method with a secondtype flow connector connecting the MAG outlet and UFL entity fluidinlet.

FIG. 46C illustrates third type sample processing method with a thirdtype flow connector connecting the MAG outlet and UFL entity fluidinlet.

FIG. 47 illustrates fourth type sample processing method includingmultiple UFLs, a fourth type flow connector, and multiple MAGs.

FIG. 48 illustrates fifth type sample processing method includingmultiple UFLs, a fifth type flow connector, and multiple MAGs.

FIG. 49 illustrates fifth type sample processing method includingmultiple UFLs, a sixth type flow connector, and multiple MAGs.

FIG. 50 illustrates seventh type sample processing method includingmultiple MAGs, a fourth type flow connector, and multiple UFLs.

FIG. 51 illustrates eighth type sample processing method includingmultiple MAGs, a fifth type flow connector, and multiple UFLs.

FIG. 52 illustrates ninth type sample processing method includingmultiple MAGs, a sixth type flow connector, and multiple UFLs.

FIG. 53 illustrates tenth type sample processing method including one ormore of UFLs and MAGs, a fifth or a sixth type flow connector, anddifferent type of cell processing devices.

FIG. 54A illustrates eleventh type sample processing method including amulti-stage MAG process.

FIG. 54B illustrates twelfth type sample processing method including amulti-cycle MAG process.

FIG. 54C illustrates thirteenth type sample processing method includinga multi-stage UFL process.

FIG. 55A illustrates first example of closed and disposable fluidiclines for third type sample processing method.

FIG. 55B illustrates fluidic lines of FIG. 55A being connected to, orattached with, various fluidic devices to realize third type sampleprocessing method.

FIG. 56A illustrates second example of closed and disposable fluidiclines for third type sample processing method.

FIG. 56B illustrates fluidic lines of FIG. 56A being connected to, orattached with, various fluidic devices to realize third type sampleprocessing method.

FIG. 57A illustrates example of closed and disposable fluidic lines forfirst type sample processing method.

FIG. 57B illustrates fluidic lines of FIG. 57A being connected to, orattached with, various fluidic devices to realize first type sampleprocessing method.

FIG. 58A illustrates example of closed and disposable fluidic lines forsecond type sample processing method.

FIG. 58B illustrates fluidic lines of FIG. 58A being connected to, orattached with, various fluidic devices to realize second type sampleprocessing method.

FIG. 59A illustrates example of closed and disposable fluidic lines forsample processing through a single MAG.

FIG. 59B illustrates fluidic lines of FIG. 59A being connected to, orattached with, various fluidic devices to realize sample processingthrough a single MAG.

FIG. 60A illustrates example of closed and disposable fluidic lines forsample processing through a single UFL.

FIG. 60B illustrates fluidic lines of FIG. 60A being connected to, orattached with, various fluidic devices to realize sample processingthrough a single UFL.

FIG. 61A illustrates replacing peristaltic pumps of FIG. 56B with usingpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 61B illustrates replacing peristaltic pumps of FIG. 56B with usingvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 62A illustrates replacing peristaltic pumps of FIG. 57B with usingpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 62B illustrates replacing peristaltic pumps of FIG. 57B with usingvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 63A illustrates replacing peristaltic pumps of FIG. 58B with usingpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 63B illustrates replacing peristaltic pumps of FIG. 58B with usingvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 64A illustrates replacing peristaltic pumps of FIG. 59B with usingpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 64B illustrates replacing peristaltic pumps of FIG. 59B with usingvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 65A illustrates replacing peristaltic pumps of FIG. 60B with usingpressurized chambers on input sample bags to drive fluid through fluidiclines.

FIG. 65B illustrates replacing peristaltic pumps of FIG. 60B with usingvacuum chambers on output sample bags to drive fluid through fluidiclines.

FIG. 66 illustrates a first process flow to separate biological entitiesfrom peripheral blood using UFL and MAG.

FIG. 67 illustrates a second process flow to separate biologicalentities from peripheral blood using MAG.

FIG. 68 illustrates a third process flow to separate biological entitiesfrom peripheral blood using MAG.

FIG. 69 illustrates a fourth process flow to separate biologicalentities from peripheral blood using MAG.

FIG. 70 illustrates a fifth process flow to separate biological entitiesfrom tissue sample using UFL and MAG.

FIG. 71 illustrates a sixth process flow to separate biological entitiesfrom tissue sample using MAG.

FIG. 72 illustrates a seventh process flow to separate biologicalentities from surface swab sample using UFL and MAG.

FIG. 73 illustrates an eighth process flow to separate biologicalentities from surface swab sample using MAG.

FIG. 74 illustrates a ninth process flow to separate biological entitiesfrom solid sample using UFL and MAG.

FIG. 75 illustrates a tenth process flow to separate biological entitiesfrom solid sample using MAG.

FIG. 76A illustrates addition of both magnetic and fluorescent labelsinto fluid samples for specific binding to target cells or entities.

FIG. 76B illustrates incubation of both magnetic and fluorescent labelsat same time to form specific binding to target cells or entities.

FIG. 77A illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL before magnetic separation by MAG.

FIG. 77B illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL after magnetic separation by MAG.

FIG. 78A illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL before magneticseparation by MAG.

FIG. 78B illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL after magneticseparation by MAG.

FIG. 79 illustrates continued process of negative MAG sample through UFLand various cell processing devices and procedures.

FIG. 80 illustrates continued process of negative MAG sample through UFLand various particle or molecule processing devices.

FIG. 81 illustrates entities analysis of negative MAG sample after MAGseparation into various analyzing devices.

FIG. 82 illustrates continued process of positive MAG sample through UFLand various cell processing devices and procedures.

FIG. 83 illustrates continued process of positive MAG sample through UFLand various particle or molecule processing devices.

FIG. 84 illustrates entities analysis of positive MAG sample after MAGseparation into various analyzing devices.

FIG. 85A illustrates adding fluorescent labels to specifically bind totarget entities within negative MAG sample immediately after negativeMAG sample collection.

FIG. 85B illustrates adding fluorescent labels to specifically bind totarget entities within positive MAG sample immediately after positiveMAG sample collection.

FIG. 86A illustrates first example of cancer treatment.

FIG. 86B illustrates second example of cancer treatment.

FIG. 86C illustrates third example of cancer treatment.

FIG. 87 illustrates first method of tumor detection.

FIG. 88 illustrates second method of tumor detection.

FIG. 89 illustrates third method of tumor detection.

FIG. 90 illustrates embodiment of first process flow to obtain cell-freeplasma.

FIG. 91 illustrates embodiment of second process flow to obtaincell-free plasma.

FIG. 92 illustrates embodiment of third process flow to obtain cell-freeplasma.

FIG. 93 illustrates method of using tumor detector for anti-agingpurpose.

For purposes of clarity and brevity, like elements and components willbear the same designations and numbering throughout the Figures, whichare not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

While the current invention may be embodied in many different forms,designs or configurations, for the purpose of promoting an understandingof the principles of the invention, reference will be made to theembodiments illustrated in the drawings and specific language will beused to describe the same. It will nevertheless be understood that nolimitation or restriction of the scope of the invention is therebyintended. Any alterations and further implementations of the principlesof the invention as described herein are contemplated as would normallyoccur to one skilled in the art to which the invention relates.

Biological entities refer to hereafter include: cell, bacteria, virus,molecule, particles including RNA and DNA, cell cluster, bacteriacluster, molecule cluster, and particle cluster. Large entities andsmall entities refer to biological entities within same fluid havingrelatively larger physical size and smaller physical size. In oneembodiment, large entities include any of: cells, bacteria, cellcluster, bacteria cluster, particle cluster, entities bound withmagnetic labels, and entities bound with optical label. In anotherembodiment, small entities include any of: molecules, particles, virus,cellular debris, non-bound free magnetic labels, and non-bound freeoptical labels. In another embodiment, large entities have a physicalsize larger than 1 micrometer (um), and small entities have a physicalsize less than 1 um. In yet another embodiment, large entities have aphysical size larger than 2 um, and small entities have a physical sizeless than 500 nanometer (nm). In yet another embodiment, large entitieshave a physical size larger than 5 um, and small entities have aphysical size less than 2 um. Biological sample include: blood, bodyfluid, tissue extracted from any part of the body, bone marrow, hair,nail, bone, tooth, liquid and solid from bodily discharge, or surfaceswab from any part of body. Entity liquid, or fluid sample, or liquidsample, or sample solution, include: biological sample in its originalliquid form, biological entities being dissolve or dispersed in a bufferliquid, or biological sample after dissociation from its originalbiological sample non-liquid form and dispersed in a buffer fluid.Biological entities and biological sample may be obtained from human oranimal. Biological entities may also be obtained from plant andenvironment including air, water and soil. Entity fluid, or fluidsample, or sample may contain various types of magnetic or opticallabels, or one or more chemical reagents that may be added duringvarious steps within the embodiments of this invention. Sample flow rateis volume amount of a fluid sample flowing through a cross-section of achannel, or a fluidic part, or a fluidic path, in a unit time, wherevolume may be in unit of liter (l), milliliter (ml), microliter (ul),nanoliter (nl), and unit time may be in unit of minute (min), second(s), millisecond (ms), microsecond (us), nanosecond (ns). Sample flowspeed is the distance of a free molecule or a free entity that travelswithin a liquid sample in a channel, or a fluidic part, or a fluidicpath, in a unit time, where distance may be in unit of meter (m),centimeter (cm), millimeter (mm), micrometer (um). Separation efficiencyis percentage of target entities within a liquid sample that aresuccessfully separated from the liquid sample by a method designed toseparate the target entities. Buffer fluid is a fluid base wherebiological entities may be dissolved into, or dispersed into, withoutintroducing additional biological entities.

FIG. 4 shows a cross-sectional view of the first embodiment of amagnetic separation device (“MAG”) of the current invention. MAG 121 iscomposed of two magnetic field producing poles, pole 102 and pole 103.Each of the poles 102 and 103 is composed of soft magnetic material,which may include one or more elements of iron (Fe), cobalt (Co), nickel(Ni), iridium (Ir), manganese (Mn), neodymium (Nd), boron (B), samarium(Sm), aluminum (Al). Pole 102 has a magnetic flux collection end 1023and a tip end 1021, where shape of the pole 102 is converging from theflux collection end 1023 towards the tip end 1021. In FIG. 4, fluxcollection end 1023 is a flat surface which is contacting, or inproximity to, the North Pole (“N”) surface of a permanent magnet 104.Permanent magnet 104 has a magnetization as shown by arrow 1041 in FIG.4 which points from the South Pole (“S”) surface of to the N surface ofthe magnet 104. Magnetization 1041 produces magnetic field in freespace, which can be described as flux lines 1046 emitting from N surfaceto returning to S surface of the magnet 104. Pole 102 flux collectionend 1023 being in contact with, or in close proximity to, the N surfaceof magnet 104 as shown in FIG. 4, due to the soft magnetic material ofpole 102, magnetic flux 1046 from the N surface of magnet 104 iscollected by the pole 102 and enters the body of the pole 102 throughflux collection end 1023. Due to the converging shape of the pole 102,the collected magnetic flux is mainly channeled within the soft magneticbody of the pole 102 and emitted from the tip end 1021 of pole 102. Saidclose proximity between flux collection end 1023 and N surface of magnet104 may be a gap distance in between surface of 1023 and N surface beingless than 1 mm. Tip end 1021 may have a much smaller surface area thanflux collection end 1023, which makes flux exiting the tip end 1021having a higher flux density than when flux 1045 is emitted by N surfaceof magnet 104, i.e. magnetic flux 1045 is concentrated and thus creatinga local high magnetic field and high field gradient around the tip end1021. It is preferred that tip end 1021 of the pole 102 is as small aspossible, for example as a convergence point, to produce largest fluxconcentration for achieving highest magnetic field. However, inpractice, due to manufacturing process, tip end 1021 may have a curvedor domed shape, which shall not affect the general concept of fluxconcentration by the tip end 1021. Pole 103 is similar to pole 102 inthat pole 103 has a larger flux collection end 1033 and a smaller tipend 1031, where flux collection end 1033 is in contact with, or in closeproximity to, S surface of permanent magnet 105. It is preferred thatpole 103 and magnet 105 are identical to pole 102 and magnet 104, butarranged as mirroring to pole 102 and magnet 104 around a center line1050. Magnet 105 magnetization 1051 is opposite to magnetization 1041 ofmagnet 104. Magnetic flux 1047 collected by flux collection end 1033from S surface of pole 103 is opposite to that of pole 102, and fluxemitted from tip end 1031 of pole 103 is opposite to that of tip end1021. Thus, between the gap of tip end 1021 and 1031, emitted flux canform closed loop and further enhance the magnetic field strength andfield gradient around the tip ends 1021 and 1031. Dashed lines 1045 areschematic of the flux emitted from tip end 1021 and returns to tip end1031. Flux lines 1045 closer to tip end 1021 and 1031 being denserindicates stronger magnetic field and larger field gradient closer tothe gap area. As shown in FIG. 4, top section of pole 102 is tilted tothe right side, while top section of pole 103 is tilted to the left.This tilted shape diverts the magnetic flux within poles 102 and 103away from bottom section of the poles and helps make the tip end 1021 ofpole 102 and tip end 1031 of pole 103 being the closest spaced featuresof the poles 102 and 103 to achieve high field in gap between tip ends1021 and 1031 with minimizing flux leakage between lower bodies of pole102 and 103. In FIG. 4, the tilted top sections of the poles 102 and 103form a triangle shape, or convex shape, top surface 1210 of the MAG 121,which will be described as “MAG wedge” 1210 of MAG 121 hereafter.Permanent magnets 104 and 105 may be composed of any of, but not limitedto, Nd, Fe, B, Co, Sm, Al, Ni, Sr, Ba, O, NdFeB, AlNiCo, SmCo, strontiumferrite (SrFeO), barium ferrite (BaFeO), cobalt ferrite (CoFeO).

FIG. 4 embodiment includes a rigid fixed shape channel 101. Channel 101has a channel wall enclosing a channel space 1013, where fluid samplemay flow through channel 101 in the channel space 1013 along the channel101 length direction that is perpendicular to the cross-section view ofFIG. 4. Channel 101 has a top surface 1012 and a bottom surface 1011.Bottom surface 1011 is formed in a shape conforming to the MAG wedgesurface 1210, such that when channel 101 is moved in direction 1014 tobe in contact with the MAG 121 poles 102 and 103, bottom surface 1011 ofchannel 101 is in contact with MAG wedge surface 1210 with no or minimalgap in between bottom surface 1011 and MAG wedge surface 1210. Topsurface 1012 of channel 101 is preferred to be conformal to bottomsurface 1011 to produce a channel space 1013 with a shape that maximizesexposure of fluid sample flowing through channel 101 to the highestmagnetic field region of the MAG wedge gap field 1045.

In FIG. 4 embodiment, poles 102 and 103, magnets 104 and 105, andchannel 101 extend in the direction perpendicular to the cross-sectionview of FIG. 4, which will be referred to as “length direction”hereafter. Fluid sample flows in the channel 101 and is contained inchannel space 1013 along the length direction. Channel 101 being a rigidand fixed shape channel, the wall thickness of channel 101 at surface1011 may be thinner than wall thickness at surface 1012, such thatchannel 101 mechanical robustness is maintained by the thicker wall atsurface 1012, and magnetic field effect on fluid sample is enhanced bythinner wall at surface 1011 allowing fluid sample being closer to theMAG wedge 1210 and tip ends 1021 and 1031. Channel 101 may be attachedto a non-magnetic channel holder 107 at the top surface 1012. Channelholder 107 may align channel 101 to MAG wedge 1210, move channel 101 toseparation position in contact with MAG 121, or lift channel 101 awayfrom MAG 121 after magnetic separation. Channel holder 107 may becomposed of any non-magnetic material including, but not limited to,metal, non-metal element, plastic, polymer, ceramic, rubber, silicon,and glass. In FIG. 4, flux collection ends 1023 and 1033 of the softmagnetic poles 102 and 103 may also be referred to as base ends 1023 and1033.

Permanent magnets as described in different embodiments of thisinvention, for example magnets 104 and 105 of FIG. 4, may each haveopposite magnetization direction to that is described in the each of thefigures and embodiments without affecting the designs, functions andprocesses of the embodiments.

FIG. 5 is the cross-sectional view of FIG. 4 first embodiment of MAGwith channel 101 in magnetic separation position. Channel 101 of FIG. 4moves along direction 1014 and comes into contact with MAG wedge 1210surface by bottom surface 1011. MAG 121 gap formed by tip ends 1021 and1031 is brought into contact with, or minimal distance to, wall ofchannel 101 and the fluid sample flowing in the channel 101. Channel 101“C” shape matching to the MAG wedge shape helps achieve largecross-sectional area of the channel space 1013 to maintain a high flowrate, and at the same time confines cells 10/30 in the fluid sampleflowing in channel 101 to a high field and high gradient region of theMAG 121 gap field as indicated by the field lines 1045. Compared toprior art of FIG. 3A and FIG. 3B, first embodiment MAG 121 does notintroduce foreign material in the channel 101 while achieving comparableor higher magnetic field and field gradient on cells 10/30 flowingthrough the channel 101. Removing of poles 102 and 103 together withmagnets 104 and 105 from channel 101 will eliminate field generationsource and avoids limitation of prior art domain related cells loss.Compared to prior art of FIG. 3C, MAG wedge of FIG. 5 being in contactwith the channel 101 wall brings highest achievable magnetic field andfield gradient to the fluid sample in the channel 101 for a moreefficient cell 10/30 separation. Channel 101 shape being conformal toMAG wedge shape allows channel 101 to have a large cross-sectionalsample flow area, meanwhile avoids the deficiency of prior art thatcells 10/30 at top end of a circular channel experiencing much lowermagnetic field than at lower end that ultimately limits sample flowrate. Thus, sample flow rate in channel 101 can be higher than prior artwhile achieving better magnetic separation efficiency.

FIG. 6 is same as FIG. 5, except biological entities, or cells 10/30 forsimplicity of description, are included to describe magnetic separationby MAG 121 from a fluid sample 6. Fluid sample 6 carrying cells 10/30 isflown through channel 101 along length direction of channel 101perpendicular to the FIG. 6 cross-sectional view. MAG 121 gap magneticfield magnetizes the SPL 2 attached to cells 10/30 and field gradientpulls cells 10/30 from the fluid 6 towards the MAG wedge to formconglomerate layer against the 1011 bottom surface of channel 101. Dueto MAG 121 design and channel 101 shape, cells 10/30 close to topsurface 1012 experience magnetic field not significantly lower thanclose to bottom surface 1011, and distance for cells 10/30 to travelfrom top surface 1012 to conglomerate on bottom surface 1011 is muchshorter than in prior art, these characteristics allow MAG 121 toresolve deficiencies of prior art.

FIG. 7 is a side view of FIG. 6 along direction 61 of FIG. 6. Fluidsample 6 carrying cells 10/30 flows from left to right in the channel101 as indicated by arrow 1010. With the MAG 121 gap field, cells 10/30are separated from fluid 6 to form conglomerate on the channel wall ofbottom surface 1011. FIG. 7 shows that majority of the cells 10/30 areseparated from liquid 6 at the earlier section of the channel 101length, as indicated by the crowded population of cells 10/30. Ascertain tail population cells 10/30 may have comparatively smaller sizeSPL 2 or fewer number of SPL 2 bound to it surface, time required forsuch tail population cells to be pulled to bottom surface 1011 is longerthan nominal population during fluid 6 flowing through channel 101.Thus, population of separated cells 10/30 will show density decreasefrom inlet towards outlet of channel 101.

FIG. 8A is a cross-sectional view of a second embodiment of MAG ofcurrent invention. MAG 122 in FIG. 8A is substantially similar as MAG121, except a soft magnetic shield 106 is attached to the S surface ofmagnetic 104 and N surface of magnet 105. Magnetic flux from S surfaceof magnet 104 and N surface of magnet 105 forms closure path within thesoft magnetic shield 106. MAG 122 compared to MAG 121 will have lessmagnetic flux leakage outside of the MAG 122 structure, where magneticflux generated by magnets 104 and 105 are mainly confined within thesoft magnetic material body of poles 102 and 103, and shield 106. MAG122 is preferred in applications where magnetic interference from MAG122 to other surrounding instrument or equipment is desired to beminimized.

FIG. 8B is a cross-sectional view of a third embodiment of MAG ofcurrent invention. Compared to MAG 121, MAG 123 of FIG. 8B incorporatesonly one permanent magnet 108, which is attached to both poles 102 and103, where flux from N surface of magnet 108 and flux from S surface ofmagnetic 108 is conducted by poles 102 and 103 to produce MAG 123 gapfield by tip ends 1021 and 1031. Compared to MAG 121, magnetic fluxgenerated by magnet 108 is mainly confined within the soft magneticmaterial body of poles 102 and 103, and MAG 123 is comparatively easierto assemble and produces less magnetic flux leakage.

FIG. 9 shows a cross-sectional view of the first embodiment MAG 121being used for magnetic separation in combination with a flexiblechannel 201. FIG. 9 is similar to FIG. 4, except that the rigid channel101 is replaced with a flexible channel 201. Flexible channel 201 mayassume any shape, including a circular shape tubing form, at itsnon-deformed state, but can be deformed into other shapes by externalforce. Wall material of channel 201 is deformable and may be composed ofany of, but not limited to, silicone, silicone rubber, rubber, PTFE,FEP, PFA, BPT, Vinyl, Polyimide, ADCF, PVC, HDPE, PEEK, LDPE,Polypropylene, polymer, thin metal or fiber mesh coated with polymerlayer, Flexible channel 201 is also shown in FIG. 9 to have a channelholder 107 attached to the back of channel 201. Channel holder 107 maybe composed of any non-magnetic material including, but not limited to,metal, non-metal element, plastic, polymer, ceramic, rubber, silicon,and glass. Channel 201 may attach to holder 107 through surface bonding,for example by gluing or injection molding, or via mechanical attachmentthrough components 1074 of FIG. 32. Holder 107 has a bottom surface 1070in contact with the top surface of channel 201, where surface 1070 beingpreferred to be substantially conformal to the MAG 121 wedge shape. InFIG. 9, holder 107 aligns attached flexible channel 201 to MAG 121 wedgegap and moves channel 201 towards MAG wedge gap in direction 1014.

FIG. 10 illustrates the flexible channel 201 being pushed against theMAG wedge of MAG 121 by the channel holder 107. With pressure exerted bythe holder 107 on flexible channel 201 against the MAG wedge of MAG 121,channel 201 is deformed in FIG. 10 with bottom surface 2013 of channel201 becoming conformal and in surface contact to MAG wedge surface 1210.Meanwhile, as holder 107 bottom surface 1070 may also be conformal tothe MAG wedge shape, top surface 2012 of channel may also be forced intoa substantially conformal shape to the MAG wedge. FIG. 10 depicts the“separation position” of flexible channel 201 relative to the MAG 121during magnetic separation of cells 10/30 from sample fluid 6. Shape offlexible channel 201 is substantially similar to channel 101 of FIG. 5and FIG. 6, except such shape of channel 201 at separation position isresult of channel 201 self-aligning and self-conforming to MAG wedgewithout the need of a manufacturing process to achieve shape of channel101. Additionally, the flow space within channel 201 at separationposition may be adjusted to allow for larger or smaller cross-sectionalarea of the flow space of channel 201, such that optimization of fluidsample 6 flow rate through channel 201 and cells 10/30 magneticseparation efficiency may be optimized. The flow space adjustment may beachieved by changing the vertical distance 1071 from the holder 107surface 1070 top point in contact with channel 201 top surface 2012, totip ends 1021 and 1031 or to an imaginary plane where tip ends 1021 and1031 reside. With a larger 1071 distance, flexible channel 201 is lessdeformed and a larger flow space is realized, which allows for a slowerflow speed at the same fluid flow rate. While with a smaller 1071distance, flexible channel 201 has a smaller flow space but top edge2012 is also closer to the MAG wedge gap and tip ends 1021 and 1031,which allows for higher magnetic field and faster separation of cells10/30. Thus optimization between flow rate and separation efficiency maybe achieved with adjusting the distance 1071 for a given combination ofMAG 121 design and flexible channel 201. In one embodiment, distance1071 is more than 0 mm and less than or equal to 1 mm. In anotherembodiment, distance 1071 is more than 1 mm and less than or equal to 3mm. In yet another embodiment, distance 1071 is more than 3 mm and lessthan or equal to 5 mm. In yet another embodiment, distance 1071 is morethan 5 mm and less than or equal to 10 mm. In yet another embodiment,distance 1071 is more than 2 times and less than or equal to 3 times ofthe wall thickness of flexible channel 201. In yet another embodiment,distance 1071 is more than 3 times and less than or equal to 5 times ofthe wall thickness of flexible channel 201. In yet another embodiment,distance 1071 is more than 5 times and less than or equal to 10 times ofthe wall thickness of flexible channel 201. Flexible channel 201 atseparation position functions similarly to channel 101 in FIG. 6, whereFIG. 10 also shows that during magnetic separation, cells 10/30 formconglomerate along channel 201 wall of lower surface 2013 directlyopposing the MAG wedge surface 1210. Thickness of channel 201 wall atbottom surface 2013 may be thinner than channel 201 wall at top surface2012.

FIG. 11 illustrates after magnetic separation is completed in FIG. 10,the channel holder 107 moves away from the MAG 121 in direction 1015,causing the flexible channel 201 to separate from MAG wedge of MAG 121to “lifted position” and flexible channel 201 may also return to itsnon-deformed shape, for example circular tubing as shown in FIG. 11.Magnetically separated cells 10/30 in FIG. 10 may hold the conglomerateform at the bottom surface of the flexible channel 201 at liftedposition. After FIG. 11 lifted position of flexible channels 201 isreached, dissociation procedures on the cells 10/30 within the flexiblechannel 201 to break up the conglomerate may be performed, as describedin FIG. 22A through FIG. 30B. Flexible channel 201 returning tonon-deformed shape, for example circular tubing of FIG. 11, provides alarger cross-sectional area of the channel space 1013 as shown in FIG.11 than at separation position in FIG. 10. Such larger channel space1013 may be preferred for easier dissociation of cells 10/30 from theconglomerate form. Additional buffer fluid may be injected into thechannel space 1013 of channel 201 at lifted position to assist channel201 return to non-deformed shape.

MAG 121 in FIG. 9 through FIG. 11 may be replaced by MAG 122 or MAG 123without limitation on described methods and processes.

FIG. 12 illustrates cross-sectional view of the fourth embodiment of MAG124. MAG 124 has three soft magnetic poles 111, 112 and 113. Center pole111 is attached to N surface of permanent magnet 109 at a fluxcollection end 1112, similar to flux collection end 1023 of pole 102 inFIG. 4, and flux 1048 from magnet 109 N surface is conducted by pole 111soft magnetic body and then emitted from a tip end 1111, which is muchsmaller in area size than flux collection end 1112, of pole 111, andfunctions similar to tip end 1021 of FIG. 4 to produce a local highfield around tip end 1111 by concentrating the magnetic flux conductedfrom magnet 109. Side poles 112 and 113 each have a flux collection end1122 and 1132 respectively, which are attached to same top surface of asoft magnetic bottom shield 114. Bottom shield 114 is then attached to Ssurface of the permanent magnet 109. Thus the magnetic flux 1049 fromthe S surface of magnet 109 is conducted in the body of bottom shield114 and divided between poles 112 and 113 and further conducted to thetip ends 1121 and 1131 of poles 112 and 113 respectively. Tip end 1111is formed in proximity to tip ends 1121 and 1131. In one embodiment, tipend 1111 may recess from an imaginary plane where tip ends 1121 and 1131reside towards magnet 109 by an offset distance between 0 mm to 1 mm. Inanother embodiment, tip end 1111 may recess from an imaginary planewhere tip ends 1121 and 1131 reside towards magnet 109 by an offsetdistance between 1 mm to 5 mm. In yet another embodiment, tip end 1111may recess from an imaginary plane where tip ends 1121 and 1131 residetowards magnet 109 by an offset distance between 5 mm to 10 mm. Tip end1111 is preferred to be spaced equally to tip ends 1121 and 1131. Topsection of pole 112 is tilted to the right side, while top section ofpole 113 is tilted to the left, which is similar to pole 102 and pole103 of FIG. 4. Such tilting is to increase gap between the main bodiesof poles 112 and 113 to main body of pole 111 to reduce flux leakagesuch that flux concentration around tip ends 1111, 1121 and 1131 ismaximized. When flux is emitted from tip ends 1111, 1121 and 1131, sinceflux 1048 conducted by center pole 111 is opposite to the flux 1049conducted by side poles 112 and 113, the flux forms closure between tipends 1111 to 1112, and tip ends 1111 to 1131. Thus, the magnetic fluxgenerated by N and S surface of magnet 109 is conducted within bodies ofpoles 111,112, 113 and shield 114 with minimal leakage to outside of MAG124 structure. Flux density is highest around tip end 1111, with tipends 1121 and 1131 also producing high flux density, which all indicatehigh magnetic field and field gradient around tip ends 1111, 1121 and1131. Compared to MAG 121, 122 and 123, MAG 124 has the advantage ofmore efficient flux closure within the MAG 124 soft magnetic bodies withless leakage and thus higher flux density around tip end 1111 to producehigher magnetic field and field gradient in channel 301.

Channel 301 is a rigid channel similar to channel 101 of FIG. 4, and hasa fixed shape similar to a rotated “D”. Channel 301 is shown to be inmagnetic separation position in FIG. 12, where tip ends 1111, 1121 and1131 may all be in contact with the curved bottom surface 3011 of the“D” shape of channel 301, which provides highest possible magnetic fieldand field gradient that MAG 124 can produce in the channel space wherefluid sample flows in channel 301. In another embodiment, tip end 1111may be in contact with the surface 3011 and tip ends 1121 and 1131 arenot contacting surface 3011. Top surface 3012 of channel 301, in oneembodiment may be on the imaginary plane where tip ends 1121 and 1131reside, and in another embodiment top surface 3012 may be above theimaginary plane in between 0 mm to 1 mm, and in yet another embodimenttop surface 3012 may be above the imaginary plane in between 1 mm to 5mm. In one embodiment, channel 301 wall thickness at surface 3012 isthicker than wall thickness at surface 3011. Channel 301 may be attachedto a non-magnetic channel holder 110 at the top surface 3012. Channelholder 110 may align channel 201 to MAG gap of MAG 124, move channel 301to separation position in contact with MAG 124 pole 111 tip end 1111, orlift channel 301 away from MAG 124 after magnetic separation.

FIG. 13 shows a cross-sectional view of the fourth embodiment MAG 124being used for magnetic separation in combination with the flexiblechannel 201, which is same as in FIG. 9. Channel holder 110 may bedifferent shape than channel holder 107 of FIG. 9. Before magneticseparation, channel holder 110 is attached to channel 201. Channelholder 110 aligns channel 201 to MAG gap of MAG 124 which is composed oftip ends 1111, 1121 and 1131 as in FIG. 12, and moves channel 201 intothe MAG gap of MAG 124 in direction 1014.

FIG. 14 illustrates the flexible channel 201 in separation position inthe fourth embodiment MAG 124 with cells 10/30 being separated and formconglomerate around bottom and side walls of the channel 201 close tothe tip ends 1111, 1121 and 1131. In FIG. 14, flexible channel 201 isdeformed similarly as in FIG. 10 to conform to the MAG gap boundaries,which are mainly the tip ends 1111, 1121 and 1131. Shape of channel 201may be different than channel 301 at separation position due to flexiblechannel 201 conforming to the MAG gap boundaries under pressure fromholder 110. Shape of channel 201 in FIG. 14 may provide higher liquidsample flow rate with higher separation efficiency than channel 301.Distance 1071 between the lower surface 1150 of holder 110 and tip end1111 may be adjusted to optimize flow rate in channel 201. Range ofdistance 1071 is same as 1071 described in FIG. 10.

FIG. 15A illustrates cross-sectional view of the fifth embodiment MAG125. MAG 125 is same as MAG 124, except the magnet 109 and bottom shield114 of MAG 124 of FIG. 12 are removed in MAG 125. Permanent magnets 115and 116 with opposing magnetizations 1151 and 1161 are placed in betweenpoles 111 and 112, and between poles 111 and 113, respectively as shownin FIG. 15A. Magnetizations 1151 and 1161 are horizontal in FIG. 15A,which enables center pole 111 conducting N surface flux from bothmagnets 115 and 116, while side poles 112 and 113 each conducts Ssurface flux from magnet 115 and 116 respectively. Compared to MAG 124,MAG 125 may produce higher field around tip ends 1111, 1121 and 1131 dueto two magnets 115 and 116 are used. MAG 125 may also be easier toassemble than MAG 124.

FIG. 15B illustrates cross-sectional view of the sixth embodiment MAG126. MAG 126 is same as MAG 124, except the side poles 112 and 113 areeach attached to S surface of permanent magnets 1092 and 1094respectively, with magnetizations 1093 and 1095 being opposite tomagnetization 1091 of magnet 109. Bottom shield 114 is attached to bothN surface of magnet 1092 and 1094, and S surface of magnet 109, and thusforming internal flux closure in shield 114 between magnets 109, 1092and 1094. Compared to MAG 124, MAG 126 may produce higher field aroundtip ends 1111, 1121 and 1131 due to three magnets 109, 1092 and 1092 areused in MAG 126.

FIG. 15C illustrates cross-sectional view of the seventh embodiment MAG127. MAG 127 is same as MAG 126 of FIG. 15B, except the bottom shield114 is removed.

FIG. 16 illustrates two of the third embodiment MAGs 123 are used formagnetic separation on a pair of flexible channels 201. The pair offlexible channels 201 are fixed on the same channel holder 1020 in FIG.16. The top MAG 123 and bottom MAG 123 are substantially identical, withtop MAG 123 being upside down vertically. MAG wedges of the top andbottom MAGs 123 are substantially aligned with center of top and bottomchannels 201. The magnets 108 of both top and bottom MAG 123 may havesame magnetization directions, as the arrows within magnets 108 in FIG.16 indicate, such that the magnetic fields produced in the top andbottom channels 201 by the top MAG 123 and bottom MAG 123 duringmagnetic separation have same direction horizontal field component,which limits magnetic flux leakage between top MAG 123 soft magneticpoles and bottom MAG 123 soft magnetic poles.

FIG. 17 illustrates the two MAG 123 of FIG. 16 are moved into separationposition against the two flexible channels 201, which is same process asin FIG. 10. After reaching FIG. 17 separation position, fluid samplecarrying cells 10/30 may flow through the channels 201 in lengthdirection perpendicular to the cross-section view to start magneticseparation of cells 10/30 by top and bottom MAG 123. Distance 1071between the holder 1021 surface contacting the channel 201 outer edge2012 and MAG 123 tip ends 1021 and 1031, or the imaginary plane wheretip ends 1021 and 1031 reside, may be adjusted to optimize flow rate ineach of the two channels 201. Range of adjustment of distance 1071 issame as 1071 described in FIG. 10.

MAG 123 in FIG. 16 and FIG. 17 may be replaced by MAG 121 or MAG 122,and channels 201 may also be replaced with channel 101.

FIG. 18 illustrates four of the fifth embodiment MAG 125 being used formagnetic separation on four of flexible channels 201. The four flexiblechannels 201 are fixed on the same channel holder 1040 as in FIG. 18.The four MAG 125 are substantially identical. MAG gaps of the four MAG125 are substantially aligned with center of each of the correspondingflexible channels 201. The permanent magnets arrangement within each MAG125 should be identical for example center pole of each of the four MAG125 is attached to N surfaces of both magnets within each respective MAG125, and side poles of each of the four MAG 125 are attached to Ssurfaces of magnets within each MAG 125, as shown in FIG. 18. Thus,neighboring MAG 125 nearest adjacent side poles are of same magneticpolarity, and leakage from side pole to side pole between neighboringMAG 125 may be minimized or avoided. Additionally, four of MAGs used onfour of channels 201 in FIG. 18 is only shown in FIG. 18 as an exampleof multiple channel process capability with a circular channelarrangement, where channels are positioned at center of the MAG 125circular array. Fewer and more MAG 125 used on corresponding number ofchannels 201 may be achieved in FIG. 18 type circular arrangementwithout limitation. FIG. 18 multiple channel circular arrangement withMAG 125 is intrinsically more flexible than MAG 123 as in FIG. 16, astwo pole design of FIG. 16 MAG 123 may lead to magnetic flux leakagethrough the poles of neighboring MAG 123 when number of MAG 123 is morethan two.

FIG. 19 illustrates the four MAG 125 of FIG. 18 are moved intoseparation position against the four flexible channels 201, which issame process as in FIG. 14. After reaching FIG. 19 separation position,fluid sample carrying cells 10/30 may flow through the channels 201 inlength direction perpendicular to the view of FIG. 19 to start magneticseparation of cells 10/30 by the four MAG 125. Similarly as in FIG. 17,distance 1071 between the holder 1040 surface contacting the channel 201outer edge 2012 and MAG 125 center pole 111 tip end 1111 for eachchannel 201 and MAG 125 pair may be adjusted to optimize flow rate ineach of the four channels 201. Range of adjustment of distance 1071 issame as 1071 described in FIG. 10.

MAG 125 in FIG. 18 and FIG. 19 may be replaced by MAG 124, MAG 126, orMAG 127, and where channels 201 may also be replaced with channel 301.

FIG. 20A illustrates the sixth embodiment of MAG 128 with having arotated “D” shape rigid channel 320 in separation position. MAG 128 issimilar to MAG 123, except that MAG wedge of MAG 123 is modified from atriangle shape to a flat top as in MAG 128. MAG 128 pole 1022 is similarto pole 102 of MAG 123, but with a flat top surface 1042 in pole 1022instead of a tip end in pole 102. Same flat top 1052 exists on pole 1032which is similar to pole 103 of MAG 123. Due to the flat top of the MAGwedge in MAG 128, rigid channel 320 may have a flat bottom surface 1062matching to, and being in contact with, the MAG wedge flat surface inseparation position, to gain highest magnetic field and field gradientregion from MAG 128. Channel 320 may be attached to a non-magneticchannel holder 1102 at the top surface. Channel holder 1102 may alignchannel 320 to MAG wedge of MAG 128, move channel 320 to separationposition in contact with MAG 128 poles 1022 and 1032 tip ends, or liftchannel 320 away from MAG 128 after magnetic separation.

FIG. 20B illustrates the sixth embodiment MAG 128 being used on aflexible channel 201, where channel 201 is attached to channel holder1102. Channel holder 1102 moves channel 201 towards MAG wedge of MAG 128along direction 1014.

FIG. 20C illustrates the sixth embodiment MAG 128 having the flexiblechannel 201 of FIG. 20B moved into separation position with cells 10/30being separated from a liquid sample to form conglomerate at bottomsurface of channel 201 against the top flat surface of the MAG wedge ofMAG 128. Channel 201 is forced to form into a rotated “D” shape channelby holder 1102 pushing channel 201 against the flat top of MAG wedge ofMAG 128, where channel 201 shape at separation position shows similarityto channel 320 of FIG. 20A. Distance 1071 between the holder 1102 bottomsurface 1062 contacting the channel 201 top edge 2012 and MAG 128 polesurfaces 1042 and 1052 may be adjusted to optimize flow rate in channel201. Range of adjustment of distance 1071 is same as 1071 described inFIG. 10.

Magnet 108 of MAG 128 may be replaced by placement of magnets 104 and105 as in MAG 121, and by placement of magnets 104 and 105 and bottomshield 106 as in MAG 122.

FIG. 21A illustrates the seventh embodiment MAG 129 with having a “V”shape rigid channel 330 in separation position. MAG 129 is differentfrom MAG 123 in pole shape, where pole 1024 and pole 1034 of MAG 129have flux concentration tip ends 3301 and 3302 that forms a “V” shapedconcave, instead of the triangle wedge shape of the MAG 123. With the Vshape MAG concave of MAG 129, rigid channel 330 is also made into a Vshape, with the lower edges 3303 and 3304 making direct contact with thesurface of the tip ends 3301 and 3302. Additionally, channel 330 mayalso preferably have a V shape notch into the channel at the top edge3305 following the V shape of the 3303 and 3304 edges, which helpsconfine fluid sample in the V shaped channel space 3306 to flow closerto the pole surfaces 3303 and 3004 that provide higher field and fieldgradient. Channel 330 may be attached to a non-magnetic channel holder1103 at the top surface 3305. Channel holder 1103 may align channel 330to MAG concave of MAG 129, move channel 323 to separation position incontact with poles 1024 and 1034 tip ends surface, or lift channel 330away from MAG 129 after magnetic separation.

FIG. 21B illustrates the seventh embodiment MAG 129 being used with aflexible channel 201, where channel 201 is attached to channel holder1103 at the top edge of channel 201. Channel holder 1103 moves channel201 towards MAG 129 concave along direction 1014. Channel holder 1103has a triangle shape, where a convergence point of the triangle touchesthe channel 201 top edge.

FIG. 21C illustrates the seventh embodiment MAG 129 having the flexiblechannel 201 of FIG. 21B moved into separation position with cells 10/30being separated from a liquid sample to form conglomerate at bottomsurface of channel 201 against the top surfaces of the MAG concave oftip ends 3301 and 3302 of MAG 129. Channel 201 is forced to form into a“V” shape channel by holder 1103. In FIG. 21C, holder 1103 forceschannel 201 against the MAG concave of MAG 129 with the lowerconvergence point and deforms the top wall of the channel 201 downwardsto move closer to the tip ends 3301 and 3302, while the same force alsocauses lower wall of channel 201 to conform to the MAG concave of MAG129 to make contact with the tip ends 3301 and 3302 top surfaces 3303and 3304. Thus, channel 201 shape in FIG. 21C at separation positionshows V shape similar to channel 330 of FIG. 21A, which brings cells10/30 in channel space 3306 closer to high field and high gradient tipends 3301 and 3302 and tip surfaces 3303 and 3304. Vertical distance1071 between the holder 1103 bottom convergence point contacting thechannel 201 top edge 2012, and MAG 129 tip ends 3301 and 3302 or animaginary plane where tip ends 3301 and 3302 reside, may be adjusted tooptimize flow rate in channel 201. Range of adjustment of distance 1071is same as 1071 described in FIG. 10.

Magnet 108 of MAG 129 may be replaced by placement of magnets 104 and105 as in MAG 121, and by placement of magnets 104 and 105 and bottomshield 106 as in MAG 122.

From FIG. 22A through FIG. 27D, various methods to demagnetize ordissociate magnetically separated cells 10/30 from conglomerate in MAGchannel will be described. For simplicity of description, flexiblechannel 201 is used. However, channels in FIG. 22A through FIG. 27D maybe labeled as “201/101”, indicating flexible channel 201 as used fordescription may be replaced with rigid channel 101 without affecting thefunction and results of the described method. Also for the simplicity ofdescription, MAG 123 is used in FIG. 22A through FIG. 27D, while anyother MAG embodiment together with corresponding channel as described inprior figures may be used under same concepts without limitation

FIG. 22A is substantially similar to FIG. 10, where channel 201 is atseparation position and cells 10/30 have been separated by magneticfield from MAG. In FIG. 22A, MAG 123 is used instead of MAG 121 of FIG.10. Channel holder 1081 may be different from channel holder 107 of FIG.10 by having a top surface notch that allows the cells 10/30demagnetization or dissociation magnetic structure (“DMAG”), which ispermanent magnet 120 in FIG. 22A, to be able to reach closer to thechannel 201/101 to provide sufficient field to demagnetize or dissociatecells 10/30 from the conglomerate in channel 201/101. Such notch ispreferred, but may not be required. DMAG magnet 120 is positioned awayfrom MAG 123 of FIG. 22A without affecting magnetic separation of cells10/30 by MAG 123. DMAG magnet 120 magnetization is labeled as invertical direction 1201, but may also be in horizontal direction withoutcausing functional difference. Channel 201/101 position relative to theMAG 123 and DMAG 120 in FIG. 22A is “Position 1”.

FIG. 22B is similar to FIG. 11, where channel holder 1081 moves channel201/101 away from MAG 123 and come into contact with, or is in closeproximity to, DMAG magnet 120 at the top surface of holder 1081, wherethe magnet 120 may fit into the notch of holder 1081 to provide highestmagnetic field on cells 10/20 conglomerate in channel 201/101. Cells10/30 form conglomerate after magnetic separation by MAG and do notbreak free from the conglomerate automatically due to SPL 2 on cells10/30 not self-demagnetize when they are part of a conglomerate. Byremoving cells 10/30 gradually with magnetic field gradient from magnet120, for example cells 10/30 with higher magnetic moment SPL 2 thatrespond to weaker magnetic field from DMAG 120 faster, conglomerate mayreach to a critical volume that remaining cells 10/30 in theconglomerate do not see enough magneto-static field from other cells10/30 and will self-demagnetize into individual cells 10/20 due to theregained superparamagnetic nature of SPL 2. Therefore, to dissociatecells 10/30 from conglomerate, removing certain amount of cells 10/30,or breaking up the conglomerate from a continuous large piece intomultiple smaller pieces will help cells 10/30 to achieveself-demagnetization. Channel 201/101 position relative to the MAG 123and DMAG 120 in FIG. 22B is “Position 2”. Channel 201 compared tochannel 101 may have an advantage during cells 10/30 dissociation byDMAG magnet 120, as channel 201 provides a larger channel space thatallows farther separation between free cells 10/30 and fromconglomerate, or between broken-up conglomerate pieces, which helpsreduce magneto-static coupling and enhances self-demagnetization speedof SPL 2 on cells 10/30. For flexible channel 201, before Position 2 orat Position 2, it is preferred to fill the channel 201 with additionalbuffer fluid to return the channel 201 to circular shape for largerchannel space.

FIG. 22C illustrates the cells 10/30 in the channel 201/101 of FIG. 22Bbeing dissociated from conglomerate by the DMAG magnet 120 at Position2.

FIG. 22D illustrates the channel holder 1081 moves channel 201/101 fromFIG. 22C DMAG Position 2 to a position, “Position 3”, between MAG 123and DMAG magnet 120. At Position 3, combined field on the cells 10/30within channel 201/101 may be the smallest, which may help SPL 2 toself-demagnetize. Channel 201/101 may be kept at Position 3 forextensive time to allow SPL 2 and cells 10/30 to fully self-demagnetizeand conglomerate to dissociate.

For an effective break up of conglomerate, mechanical agitations may beadded to the conglomerate by the magnetic force exerted by MAG and DMAGmagnets. For example, channel holder 1081 may alternate channel 201/101between Positions 1 and 2, or Positions 2 and 3, or Positions 1, 2 and3, such that alternating magnetic force by MAG and DMAG may move wholeor part of the conglomerate in the channel space, thus helping break upthe conglomerate into smaller pieces or cause enough cells 10/30 tobreak free from the conglomerate and conglomerate may self-dissociate.After conglomerate is sufficiently dissociated, free cells 10/30 may beflushed out of channel 201/101 at Position 3 or Position 2.

FIG. 23A illustrates mechanical vibration may be applied to the channelholder 1081 by a motor 130 when channel 201/101 is at Position 2 orPosition 3 of FIG. 22B and FIG. 22C. Such vibration may be transferredfrom holder 1081 through wall of channel 201/101 and into the fluidwithin the channel 201/101 to cause localized turbulence flow at variouslocations within the channel 201/101, which may help mechanically breakup the conglomerate into small pieces to assist conglomeratedissociation.

FIG. 23B illustrates ultrasound vibration by a piezoelectric transducer(“PZT”) 131 may be applied to the channel holder 1081. Similar to FIG.23A, ultrasound vibration may be transferred into the fluid within thechannel 201/101 to cause localized high frequency turbulence within thechannel 201/101, which may help mechanically break up the conglomerateinto small pieces to assist conglomerate dissociation.

FIG. 23C illustrates mechanical vibration of FIG. 23A may be applied tothe channel 201/101 wall directly by motor 130.

FIG. 23D illustrates ultrasound vibration of FIG. 23B may be applied tothe channel 201/101 wall directly by PZT 131.

FIG. 23E is a side view of the channel 201/101 along the direction 61 asin FIG. 22D. Arrow 1030 represents alternating direction pulsed fluidflow may be applied to the channel liquid sample to produce a flowjittering in the liquid within the channel 201/101, which may alsoproduce local turbulence flow with fluid in channel 201/101 to helpmechanically break up the conglomerate into small pieces to assistconglomerate self-dissociation. FIG. 23E alternating pulsed flow may becombined with FIG. 23A through FIG. 23D vibration methods to apply tochannel 201/101 at Position 2 or Position 3 of FIG. 22B through FIG.22D.

When conglomerate in channel 201/101 is of large size, multiple roundsof cells 10/30 dissociation with FIG. 22B to FIG. 23E methods, andflushing of cells 10/30 out of channel 201/101, may be used. During eachflush, a certain part of cells 10/30 may be washed out of channel,making dissociation of remaining cells 10/30 still in the conglomeratein channel 201/101 easier in next round.

FIG. 24A is similar to FIG. 22B, where channel holder 1081 is incontact, or in close proximity to, DMAG magnet 120 after cells 10/30 aremagnetically separated by MAG 123. Different than in FIG. 22B, DMAGmagnet 120 of FIG. 24A is positioned on the side of and away from MAG123, and holder 1081 is also rotated compared to FIG. 22B to fit its topsurface notch to the magnet 120. Placement of magnet 120 in FIG. 24 mayreduce magnetic field interference between MAG 123 and DMAG magnet 120.Channel 201/101 position relative to the MAG 123 and DMAG 120 in FIG.24A is “Position 12”.

FIG. 24B illustrates that after cells 10/30 are dissociated at Position12 of FIG. 24A, the channel 201/101 together with channel holder 1081 ofFIG. 24A are rotated away from magnet 120 of FIG. 24A into a positionbetween MAG 123 and DMAG magnet 120, where combined magnetic field fromMAG 123 and DMAG magnet 120 on channel 201/101 and cells 10/30 thereinis lowest, which is similar to Position 3 of FIG. 22D. Channel 201/101position relative to the MAG 123 and DMAG 120 in FIG. 24B is “Position13”.

FIG. 25A illustrates DMAG structure that is same as in FIG. 22B, whereDMAG structure includes only permanent magnet 120 with magnetization1201.

FIG. 25B illustrates DMAG structure that includes permanent magnet 120and a soft magnetic pole 1202 with convergence shape towards channel201/101. Soft magnetic pole 1202 convergence shape helps concentratemagnetic flux from magnet 120 to produce higher field and high fieldgradient on cells 10/30 in channel 201/101 at Position 2 to moreeffectively demagnetize and dissociate the conglomerate of cells 10/30.

FIG. 25C illustrates DMAG structure that includes permanent magnet 120and a pair of soft magnetic poles 1203 and 1204. Magnetization 1201 ofmagnet 120 is in horizontal direction, and each of poles 1203 and 1204has an convergence shape pointing towards channel 201/101, where theconvergence ends of poles 1203 and 1204 form a DMAG gap sitting in, orin close proximity to, the channel holder 1081 top surface notch, whereflux from magnet 120 is conducted by the poles 1203 and 1204 andconcentrated in the DMAG gap to produce high field and high fieldgradient on cells 10/30 in channel 201/101 at Position 2 to moreeffectively demagnetize and dissociate the conglomerate of cells 10/30.

FIG. 25D illustrates DMAG structure that includes an electromagnetincluding a soft magnetic core 1205 and coils 1206, where electriccurrent following in the coils 1206 may produce magnetization in core1205 in directions of 1207, and core 1205 functions like magnet 120 toproduct magnetic field on cells 10/30 in channel 201/101 at Position 2to demagnetize or dissociate the conglomerate of cells 10/30. Bychanging the electric current amplitude and direction in coils 1206,magnetic field from core 1205 on cells 10/30 may change strength anddirection. In one embodiment, DC current is applied to coils 1206. Inanother embodiment, AC current with alternating polarities is applied tocoils 1206. In yet another embodiment, current applied to coils 1206 isprogrammed to vary in amplitude, or in direction, or in frequency, or inamplitude ramp up or ramp down rates, to more effectively demagnetizeand dissociate the conglomerate of cells 10/30.

FIG. 25E illustrates that motor 130 as shown in FIG. 23A may producemechanical vibrations on DMAG structure of FIG. 25C, such vibrations maytransfer from DMAG structure to holder 1081 through DMAG structure toholder 1081 contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structuresdescribed in FIG. 25A through FIG. 25D.

FIG. 25F illustrates that PAT 131 as shown in FIG. 23B may produceultrasound vibrations on DMAG structure of FIG. 25C, such vibrations maytransfer from DMAG structure to holder 1081 through DMAG structure toholder 1081 contact, and finally transferred to fluid in channel201/101, where DMAG structure can be changed to any of DMAG structuresdescribed in FIG. 25A through FIG. 25D.

To achieve demagnetization and dissociation of cells 10/30 fromconglomerate in channel 201/101, an alternative method as described inFIG. 26A through FIG. 26D may be used without using a DMAG structure,where function of DMAG structure is achieved with same MAG.

FIG. 26A is same as FIG. 22A, where channel 201/101 is at separationposition and cells 10/30 are separated by magnetic field of MAG 123 inchannel 201/101, except channel holder 1082 may not have the top surfacenotch as holder 1081. Channel 201/101 position relative to the MAG 123in FIG. 26A is “Position 21”.

FIG. 26B illustrates channel 201/101 of FIG. 26A is lifted from MAG 123to a lower field position, “Position 22”. At Position 22 channel 201/101may rotate around its center as indicated by arrow 210, preferable by180 degrees. Such rotation may require channel 201/101 not beingpermanently fixed to holder 1082

FIG. 26C illustrates channel 201/101 of FIG. 26B after rotation of 180degrees at Position 22, the cells 10/30 conglomerate formed on innerwall of channel 201/101 rotates together with channel wall to be at thetop end of the channel 201/101 relative to MAG 123.

FIG. 26D illustrates that channel 201/101 is moved from Position 22closer to MAG 123 to a Position 23 in between Position 21 and Position22, where magnetic field from MAG 123 on cells 12/30 is stronger thanPosition 22 but weaker than Position 21. Cells 10/30 in conglomerate attop end of channel 201/101 may then be pulled away by MAG 123 field fromconglomerate and demagnetization and dissociation of conglomerate maystart. The process of FIG. 26B through FIG. 26D may repeat multipletimes, where channel 201/101 may return to Position 22 from Position 23to perform another rotation and then move back to Position 23, untilcells 10/30 are sufficiently dissociated in channel 201/101. At end ofdemagnetization, cells 10/30 may be flushed out of channel 201/101preferably at Position 22. Mechanical vibrations and flow jittering asdescribed in FIG. 23C through FIG. 23E may be applied to channel 201/101at Position 22 and Position 23.

FIG. 27A is same as FIG. 26A, where channel 201/101 is at separationposition and cells 10/30 are separated by magnetic field of MAG 123.Channel 201/101 position relative to the MAG 123 is “Position 21”.Channel 201/101 is attached to holder 1082 in FIG. 27A.

FIG. 27B illustrates channel 201/101 of FIG. 27A is lifted from MAG 123to lower field Position 22 by holder 1082.

FIG. 27C illustrates that at Position 22, dissociation of cells 10/30 inchannel 201/101 may be achieved only through mechanical vibrationexerted by motor 130. FIG. 27C shows that motor 130 applies mechanicalvibration to holder 1082, where such vibration may be transferred fromholder 1082 through wall of channel 201/101 and into the fluid withinthe channel 201/101 to cause localized turbulence flow at variouslocations within the channel 201/101, which may help mechanically breakup the conglomerate into small pieces to assist self-dissociation ofcells 10/30 conglomerate. Motor 130 may also exert vibration directly onchannel 201/101 as shown in FIG. 23C instead of through holder 1082.Alternating direction pulsed fluid flow as described in FIG. 23E may beapplied to the channel liquid sample to produce a flow jittering in theliquid within the channel 201/101 at the same time of motor 130vibration application.

FIG. 27D illustrates that at Position 22, dissociation of cells 10/30 inchannel 201/101 may be achieved primarily through ultrasound vibrationexerted by PZT 131. FIG. 27D shows that PZT 131 applies ultrasoundvibration to holder 1082, where the ultrasound vibration may betransferred into the fluid within the channel 201/101 to cause localizedhigh frequency turbulence within the channel 201/101, which may helpmechanically break up the conglomerate into small pieces to assistself-dissociation of cells 10/30 conglomerate. PZT 131 may also exertultrasound vibration directly on channel 201/101 as shown in FIG. 23D.Alternating direction pulsed fluid flow as described in FIG. 23E may beapplied to the channel liquid sample to produce a flow jittering in theliquid within the channel 201/101 at the same time of PZT 131 ultrasoundvibration application.

FIG. 28A through FIG. 30B describe embodiments of methods to assistcells 10/30 conglomerate dissociation by mechanical agitations, whichmay be applied to channel 201/101 as in FIG. 27B at Position 22 andapplied to channel 201/101 as in FIG. 22B through FIG. 22D, FIG. 23Athrough FIG. 23D, FIG. 24A through FIG. 25F, FIG. 26B through FIG. 26D,FIG. 27B through FIG. 27D.

FIG. 28A shows a side view of channel 201/101 and holder 1082 alongdirection 61 of FIG. 27B, where cells 10/30 are magnetically separatedby MAG field and form conglomerate on lower side of the channel 201/101wall. Channel mounts 1073 may be used to attach channel 201/101 tochannel holder 1082. Channel mounts 1073 may fix channel 201/101 atsections attached to mounts 1073 as anchors against channel 201/101deformation, compression or elongation during mechanical agitationprocess. Channel mounts 1073 may also perform a valve function thatcloses fluid flow into or out of flexible channel 201 section betweentwo channel mounts 1073 before mechanical agitation process of FIG. 28A,such that fluid enclosed in channel 201 may more efficiently producelocalized turbulence within the channel 201. FIG. 28A illustrates thatan externally applied force 300 may stretch or deform the channel201/101 in a direction away from the holder 1082, for exampleperpendicular to the channel 201/101 length direction. Such deformationor stretch of channel 201/101 builds up elastic energy in the channel201/101 wall material.

FIG. 28B illustrates that force 300 of FIG. 28A is released, and elasticenergy built up in channel 201/101 wall acts to spring back channel201/101 towards its original non-deform and non-stretch position.Depending on channel 201/101 wall material property, such spring backmay provide a transient turbulence flow at various locations within thechannel 201/101, which may help mechanically break up the cells 10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30 conglomerate. After release of force 300 and channel 201/101spring back, alternating flow 1030 may be applied similarly as in FIG.23E to assist dissociation process of conglomerate of cells 10/30, wherevalve function of 1073 may turn off to allow fluid flow within channel201/101.

The FIG. 28A and FIG. 28B channel 201/101 deform/stretch and releaseprocess may be repeated as many times as needed until conglomerate ofcells 10/30 are sufficiently dissociated, which may then be flushed outof channel 201/101 by buffer fluid.

FIG. 29A illustrates an alternative method of mechanical agitation fromFIG. 28A. Every aspect is same as in FIG. 28A, except that a compressiveforce 302 may be applied to compress channel 201 in directionperpendicular to the channel 201 length direction, for examplecompressing channel 201 against channel holder 1082 as shown in FIG.29A. As liquid within channel 201 has limited compressibility, force 302may cause channel 201/101 wall to expand in direction perpendicular tothe view of FIG. 29A, i.e. in direction perpendicular to both channellength direction and force 302 direction. Such expansion of channel201/101 wall will again build up elastic energy in the channel 201 wallmaterial.

FIG. 29B is same as FIG. 28B in every aspect, except that FIG. 29B isafter compressive force 302 of FIG. 29A is released, and elastic energybuilt up in channel 201 wall acts to spring back channel 201 to itsoriginal non-compressed shape. Such spring back may provide a strongtransient turbulence flow at various locations within the channel 201,which may help mechanically break up the cells 10/30 conglomerate intosmaller pieces to assist self-dissociation of cells 10/30 conglomerate.After release of force 302 and channel 201 shape spring back,alternating flow 1030 may be applied similarly as in FIG. 23E to assistdissociation process of conglomerate of cells 10/30, where valvefunction of 1073 may turn off.

The FIG. 29A and FIG. 29B channel 201 compression and release processmay be repeated as many times as needed until conglomerate of cells10/30 are sufficiently dissociated, which may then be flushed out ofchannel 201.

FIG. 30A illustrates another alternative method of mechanical agitation.Every aspect is same as in FIG. 28A, except that rotational twistingforce 303 or 304 may be applied to channel 201 to twist channel 201along channel length direction, as shown in FIG. 30A. In one embodiment,only one of rotational force 303 or 304 is applied to one end of channel201. In another embodiment, both rotational force 303 and force 304 areapplied to difference ends of the channel 201 in opposite rotationaldirections to cause the channel 201 to twist along channel lengthdirection. Such twist deformation of channel 201 will again build upelastic energy in the channel 201 wall material.

FIG. 30B is same as FIG. 28B in every aspect, except that FIG. 30B isafter rotational force 303 and force 304 of FIG. 30A are released, andelastic energy built up in channel 201 wall acts to spring back channel201 towards its original non-twisted shape. Such spring back may providea strong transient turbulence flow at various locations within thechannel 201, which may help mechanically break up the cells 10/30conglomerate into smaller pieces to assist self-dissociation of cells10/30 conglomerate. After release of forces 303 and 304, and channel 201shape spring back, alternating flow 1030 may be applied similarly as inFIG. 23E to assist dissociation process of conglomerate of cells 10/30,where valve function of 1073 may turn off.

The FIG. 30A and FIG. 30B channel 201 twist and release process may berepeated as many times as needed until conglomerate of cells 10/30 aresufficiently dissociated, which may then be flushed out of channel 201by buffer fluid.

Mechanical forces 300, 302, 303 and 304 may be applied by mechanicalstructures that are motorized and able to apply such forces repeatedlyto channel 201, examples may include a flap for providing force 300, acompressor for provide force 302, and twisters for providing forces 303and 304.

FIG. 31 is a schematic diagram illustrating a method to use MAG toseparate biological entities conjugated with magnetic labels, forexample cells 10/30, from a fluid solution. MAG channel of FIG. 31 maybe any of channels 101, 201, 301, 320, or 330 described in any of thefigures of this specification, and MAG of FIG. 31 may be any of the MAG121, 122, 123, 124, 125, 126, 127, 128, or 129 described with thecorresponding channel in any of the said figures. Method of FIG. 31 mayinclude the following steps in sequence. In step 400, MAG channel ispositioned with its outside wall contacting MAG wedge surface or poletip ends, i.e. separation position of Position 1 or Position 21 as inFIG. 5, FIG. 10, FIG. 12, FIG. 14, FIG. 17, FIG. 19, FIG. 20A, FIG. 20C,FIG. 21A, FIG. 21C, FIG. 22A, FIG. 26A, FIG. 27A. In step 401, fluidsample is flown through the MAG channel in separation position. Then instep 402, positive entities with magnetic labels SPL 2 attached, forexample cells 10/30, and free magnetic labels SPL 2 within the fluidsample are attracted by the magnetic field of MAG and agglomerate at theMAG channel wall against the MAG wedge or MAG pole tip ends. Meanwhile,in step 4020, negative entities without magnetic labels SPL 2 attachedpass through the MAG channel without being attracted. The negativeentities may then be processed directly in subsequent procedures asshown by path 427, where subsequent procedures may include entitiesanalysis 407, for example processes as included in FIG. 79 through FIG.81, or negative entities may be passed for continued process 408, forexample through a UFL device as shown in FIG. 46A through FIG. 46C, FIG.50 through FIG. 52, or through repeated MAG process as in FIG. 54A andFIG. 54B. After step 402, in step 403, sample may be depleted at inputof the MAG channel and magnetic separation of positive entities may becompleted. In step 404, which is an optional step, buffer fluid may beflown through MAG channel with MAG channel still at separation positionto wash off any negative entities without magnetic labels SPL 2 but mayhave resided with the conglomerate of positive entities due tonon-specific bindings. Then in step 405, MAG channel may be moved awayfrom MAG to dissociation position including Position 2 and Position 22at in FIG. 11, FIG. 22B, FIG. 22D, FIG. 24B, FIG. 26B, FIG. 26D, FIG.27B, and magnetic dissociation 451, as shown in FIG. 22A through FIG.26D, or mechanical dissociation 452 as shown in FIG. 27C through FIG.30B, or magnetic together with mechanical dissociate 453 may be appliedto the positive entities in MAG channel. In step 406, buffer fluid maybe flown through MAG channel to flush out dissociated positive entities.If positive entities are not completely dissociated, 465 shows thatrepeated dissociation process 405 may be applied to remaining positiveentities in MAG channel after prior flush out step, until positiveentities are sufficiently dissociated and flushed out of the MAGchannel. In the case that fluid sample is in large volume, fluid samplemay be separated into multiple sub-volumes, where after process of asub-volume from step 400 to step 406, a next sub-volume may be inputinto the MAG channel starting from step 400 for continued process asshown by 461 until completion of the fluid sample of the large volume.After positive entities are collected after step 406, they may beprocessed in subsequent procedures as shown by path 428, wheresubsequent procedures may include entities analysis 407 or continuedprocess 408.

FIG. 32 illustrates a method to align channel 201/101 to MAG gap of MAG123 device. Precise alignment of channel 201/101 to MAG wedge or MAGpole tip ends is of importance as descried in embodiments of thisinvention. In FIG. 32, side fixtures 1074 may be used to align andposition channel 201/101 to designated locations on channel holder 1081or 1082, where the fixtures 1074 may be fit into a pre-defined slot,notch, clip or other physical features on the sides of the channelholder 1081/1082. In one embodiment, channel 201/101 may be slightlystretched in channel length direction, thus channel 201/101 may have areduced width 2011 in between the fixtures 1074, where such stretchhelps guarantee a straight channel which may be then aligned with astraight MAG wedge of MAG 123. After channel 201/101 is attached toholder 1081/1082 by fixtures 1074, holder 1081/1082 may then movechannel 201/101 to separation position, where holder 1081/1082 may havepre-determined physical orientation to MAG 123, for example a hinge,which aligns channel 201/101 to MAG wedge or MAG pole tip ends of MAG123 precisely. Fixtures 1074 may be the same as 1073 as in FIG. 28Athrough FIG. 30B.

FIG. 33A through FIG. 37 illustrate method to utilize peristaltic pumpsin embodiments of this invention.

FIG. 33A illustrates a typical peristaltic pump 500, which includes arotor 501, drivers 502 attached to the rotor 501, and pump tubing504/505, where tubing 504 is fluid incoming section and tubing 505 isfluid outgoing section of the same pump tubing. When rotor 501 rotatesin direction 503, drivers 502 will squeeze pump tubing and force fluidto move from incoming section 504 to outgoing section 505 in directions5041 and 5051 respectively. In the case when rotor rotates reversely todirection 503, fluid moves from outgoing section 505 to incoming section504 of the pump tubing. Connectors 506 and 507 may be optionalconnections to incoming fluid line and outgoing fluid line 508respectively. Advantage for peristaltic pump is the tubing 504/505 maybe included as a continuous part of an enclosed fluid line as shown inFIG. 55A through FIG. 60B, which may be made disposable and single use,as well as sterile for clinical purpose. However, due to the spaceddrivers 502 along the circumference of the rotor 501, flow rate of fluidoutput from section 505 has pulsation behavior, where flow rateincreases and decreases with moving of each of the driver 502. Suchpulsation is not desired for MAG and UFL fluid driving. FIG. 33A showsoutput section 505 outputs fluid through connector to channel 508.Channel 508 is preferred to be flexible tubing. Channel 508 may also bea section of channel 201. Flow limiter parts 509 and 510 functiontogether to effectively clamp onto the channel 508 to reduce the fluidflow rate passing through the limiter. With reduced flow rate throughthe limiter, continued fluid output from pump 500 section 505 into thechannel 508 will build up fluid pressure within channel 508. Due to theflexible nature of channel 508, channel 508 may enlarge its widthperpendicular to the channel length direction, and forms fluid reservoirwithin channel 508 with elastic stress built up in channel wall. Duringpulsation of output flow from pump 500, when 5051 flow rate increases,channel 508 width will increase to build up stress in 508 channel walland pressure within channel 508 and increase volume of channel 508absorbs most of the instantaneous incoming flow, while flow rate 520through limiter 509/510 into channel 501 shows smaller increase. When5051 flow rate decreases, built-in elastic stress in channel 508 walland fluid pressure in channel 508 continues to push fluid through thelimiter 509/510, and flow rate 520 shows smaller flow rate decrease.

FIG. 33B illustrates top-down view of the inner structure of first typeflow limiter 509 along the direction 63. FIG. 33B shows that flowlimiter 509 has a shaped trench 5011, which allows fluid to flow throughchannel 508 when limiters 509 and 510 clamp onto channel 508 as in FIG.33A. Trench 5011 has entrance width 511 to incoming fluid and exit width512 to channel 201, where width 511 may be larger than width 512.Decreased trench 5011 width from 511 to 512 reduces the flow ratethrough the limiter 509/510. Flow limiter 510 may have same top downview and structure as limiter 509 when view in direction opposite to 63.

FIG. 33C illustrates a second type flow limiter in same view as FIG.33A, where after flow limiters 509 and 510 clamp onto channel 508, flowlimiters 509/510 form an effective opening of 514 towards channel 508,and opening of 513 towards channel 201. Opening 513 may be smaller thanopening 514, which reduces flow rate through the limiter 509/510.

FIG. 34A is same as FIG. 33A except flow limiters 509/510 are disengagedfrom the flexible channel 508, where flow from pump 500 through channel508 and channel 201 is continuous without limiter 509/510 and there isno elastic stress built up in channel 508 wall.

FIG. 34B is a schematic illustration of fluid flow rate 520 as in FIG.34A situation, which shows large pulsation in flow rate 520. FIG. 34Bshows the example 520 flow rate value vs pump 500 operation time frompumping start to pumping end. Value 521 illustrates the high flow rateand value 522 illustrates low flow rate of the pulsation behavior.

FIG. 35A is same as FIG. 33A, where flow limiters 509/510 are clampedupon flow channel 508, flow rate through the flow limiters 509/510 isreduced, and channel 508 has enlarged channel width with elastic stressbuilt up in channel 508 wall.

FIG. 35B is a schematic illustration of fluid flow rate 520 as in FIG.35A situation, which shows pulsation reduction in flow rate 520 comparedto FIG. 34B. Value 523 corresponds to value 521 of FIG. 34B, and value524 corresponds to value 522 of FIG. 34B. FIG. 35B illustrates thatlimiters 509/510 effectively reduce 520 flow rate pulsation. Due to thechannel 508 liquid pressure build up at the start of pumping, andchannel 508 liquid pressure dissipation at end of pumping, whilelimiters 509 and 510 are engaged, a flow rate ramp up slope 5221 afterpump start and flow rate ramp down slope 5222 after pump end may existin FIG. 35B.

FIG. 36A and FIG. 36B illustrate method to use flow limiter 509/510 togenerate instantaneous high flow rate short pulse through channel 201for flushing out magnetically separated entities, for exampledissociated cells 10/30.

FIG. 36A illustrates FIG. 33A and FIG. 35A situation, where flowlimiters 509 and 510 are clamped onto the flexible channel 508 whilepump 500 pumps fluid into channel 508, where pressure is built up withinthe flexible channel 508, and elastic stress is built up in wall ofchannel 508. Line 525 represents a continuous channel 201 from after thelimiters 509/510 to channel 201 over MAG structure. Flow rate 5201represents averaged flow rate of flow rates 523 and 524 of FIG. 35B whenflow limiters 509 and 510 are engaged.

FIG. 36B illustrates that flow limiters 509 and 510 are disengaged fromthe flexible channel 508, similar to FIG. 34A situation, while pump 500still pumps fluid into channel 508, or immediately after pump 500 stopspumping and before pressure within channel 508 dissipates. Atdisengagement of limiters 509 and 510, liquid pressure in channel 508and elastic stress in wall of channel 508 produces an instant high speedfluid pulse flow 5202 into channel 201 which may flush the magneticallyseparated entities out of the channel 201. Such high speed short pulseflow 5202 may help achieve complete flush out of cells 10/30 with smallvolume of fluid that is originally contained in channel 508 of FIG. 36A.FIG. 36B also shows that a rigid cladding structure 1075 may be put intocontact with channel 201 to help reduce deformation of flexible channel201 during the cells 10/30 flush out to maintain the flow speed inchannel 201.

FIG. 37 is a schematic illustration of fluid flow rate pulse created bythe flow limiter operation of FIG. 36A to FIG. 36B, where 5201 is thefluid flow rate in channel 201 before limiters 509 and 510 release, and5202 is flow rate peak value after limiters 509 and 510 release.

From FIG. 33A through FIG. 36B, channel 508 is a flexible channel, whilechannel 201 may be replaced by a rigid channel 101, 301, 320, or 330.

FIG. 38A through FIG. 43 describe various embodiments of micro-fluidicchip (“UFL”) and method of use.

FIG. 38A is a top-down view of a first UFL embodiment UFL 600, wheremicro-fluidic channels are formed as trenches into a substrate material611. UFL contains an entity fluid 6020 inlet 602, a buffer fluid 6040inlet 604, a main channel 601, a large entities 6070 outlet 607, and asmall entities 6090 outlet 609. Two side channels 603 connect inlet 602to main channel 601 from the two sides of the main channel 601. Inlet604 is directly connected to the main channel 601 at the center of themain channel 601. Main channel 601 connects to outlet 607 at the centerof the main channel 601, and connects to outlet 609 from two sides ofmain channel 601 through two side channels 608. Entities fluid 6020contains both large entities 6070 and small entities 6090. Buffer fluid6040 is fluid for providing UFL function but without biologicalentities. Large entities 6070 fluid from outlet 607 contains mainlylarge entities 6070 and buffer fluid 6040. Small entities 6090 fluidfrom out 609 contains mainly small entities 6090 and fluid of entitiesfluid 6020 and may contain certain amount of buffer fluid 6040. Duringoperation of UFL 600, buffer fluid 6040 and entity fluid 6020 aresimultaneously pumped into outlets 604 and 602 respectively, wherebuffer fluid 6040 flows along center line of the main channel 601 andentity fluid flows close to the two side of the main channel as laminarflow. Buffer fluid 6040 carries large entities 6070 to exit outlet 607and entity fluid carries remaining small entities 6090 to exit outlet609. Channel 601 is substantially straight and linear along channellength direction from inlet 604 to outlet 607.

FIG. 38B is a cross-sectional view of a portion of the FIG. 38A UFL 600along direction 64, which includes entity fluid inlet 602, buffer fluidinlet 604, and part of the UFL main channel 601. FIG. 38B illustratesUFL 600 is compose of two components, substrate 611 and cover 610.Inlets 602 and 604, outlets 607 and 609, channels 601, 603 and 608 areformed in substrate 611 as trenches of same depth 627 and preferablyformed in a single step. In one embodiment, depth 627 is between 100 nmto 500 nm. In another embodiment, depth 627 is between 500 nm to 1 um.In yet another embodiment, depth 627 is between 1 um to 10 um. In yetanother embodiment, depth 627 is between 10 um to 100 um. In yet anotherembodiment, depth 627 is between 100 um to 1 mm. Cover 610 containsexternal access ports to inlets and outlets of UFL 600 to allow entitiesfluid 6020 and buffer fluid 6040 to enter inlets 602 and 604, and toallow large entities 6070 fluid and small entities 6090 fluid to exitoutlets 607 and 609. Inlets 602 and 604, outlets 607 and 609 are shownto be circular shape in FIG. 38A, but may be any other shape, includingellipse, square, rectangle, triangle, polygon, as suitable forapplication. Access ports of cover 610 are clearances, i.e. holes,through cover 610 directly over the inlets and outlets 602, 604, 607 and609. FIG. 38B shows example of access ports 621 and 641 clearancesmatching to inlets 602 and 604 positions. After manufacture of the UFL600 substrate 611 with the trenches of inlets, outlets and channels, andcover 610 with the access ports, cover 610 is positioned over thesubstrate 611 to form enclosed channels 601, 603 and 608, where cover610 may bond to substrate 611 through any of: (1) surface to surface Vander Waals force; (2) gluing; (3) ultrasound thermal melting when one orboth of substrate 611 and cover 610 being made of plastic or polymermaterial. Access ports clearances of cover 610, for example 621 and 641to inlets and outlets 602, 604, 607 and 609 are preferred to be smallerin size than the corresponding inlets and outlets, which allows forpositioning error during cover 610 to substrate 611 alignments withoutcausing function loss of UFL due to misalignment. Injectors 6021 and6041 then show example of possible external fluid injection to inlets ofUFL 611 through cover 610 access ports clearance, where the injectors6021 and 6041 may have a larger nozzles size than the matching accessports 621 and 641 for managing positioning errors between injectors andaccess ports. FIG. 38B shows that entities fluid 6020 containing largeentities 612 and small entities 613, which may be injected by injector6021, passing through assess port 621 and into inlet 602 and passinginto main channel 601 as side laminar flows, while buffer fluid 6040 maybe injected by injector 6041, passing through assess port 641 and intoinlet 604 and passing into main channel 601 as center laminar flow.

Substrate 601 may be composed of any of: glass, silicone,aluminum-titanium-carbon (AlTiC), plastic, polymer, ceramic, or metal,where metal may be composed any one or any alloy of iron, nickel,chromium, platinum, tungsten, rhenium. In one embodiment, forming ofinlets, outlets and channels in substrate 611 includes the steps of: (1)providing a substrate 611 having one substantially flat surface; (2)forming etching mask on top of said flat surface; (3) etch of substratewith a first etching method including: wet etch with fluid chemical, dryetch with chemical gas, plasma enhanced dry etch, sputter etch with ionplasma, and ion beam etch (IBE). In forming of etch mask of step (2),etch mask may be composed of photo resist (PR), which may includedeposition or spin coating of PR on said flat surface; exposure byoptical or ion/electron radiation with patterns of inlets, outlets andchannels; development of PR after said exposure, where remaining PR withsaid patterns serves as etch mask. Etch mask may also be made of a hardmask material that has lower etch rate than the substrate material underthe first etching method, and step (2) may include: deposition of a hardmask layer on said flat surface; deposition or spin coating PR layer onhard mask layer; exposure of said PR by optical or ion/electronradiation with patterns of inlets, outlets and channels, development ofPR after said optical exposure, where remaining PR with said patternserves as etching mask of said hard mask; hard mask is etched throughwith a second etch method including any of: wet etch with fluidchemical, dry etch with chemical gas, plasma enhanced dry etch, sputteretch with ion beam; removal of remaining PR layer. Second etch methodand first etch method may be different in type, or different inchemistry.

In another embodiment, inlets, outlets and channels in substrate 611 maybe formed by thermal press involving using a heated stencil withphysical pattern of the inlets, outlets and channels to melt and deformpart of substrate 611 to construct the inlets, outlets and channels,then cooling down substrate 611 and remove the stencil. In thermalpress, substrate material is preferred to be plastic or polymer. In yetanother embodiment, inlets, outlets and channels in substrate 611 may beformed by imprint, which involves using a stencil with physical patternof the inlets, outlets and channels to imprint into a partially orcompletely melt substrate 611, and then cooling the substrate 611 andfinally removing stencil, where cooled substrate retains the patterntransferred from stencil of the inlets, outlets and channels. Inimprint, substrate material is preferred to be plastic or polymer. Inanother embodiment, inlets, outlets and channels are formed in substrate611 by injection molding, where melted substrate 611 materials areinjected into a mold cavity where substrate 611 body with engravedinlets, outlet and channels are defined by the mold cavity. Cover 610may compose similar to substrate 611 materials, and assess ports ofcover 610 may be formed in cover 610 similarly as the inlets, outlet andchannels formed in substrate 611 as described above.

FIG. 38C is a schematic diagram illustrating a single fluidic pressurenode 615 created between two side walls of the UFL 600 channel 601 ofFIG. 38A by ultrasound vibration generated by a PZT 614. FIG. 38C is across-section view along direction 65 of FIG. 38A for part of the UFL600 including main channel 601, substrate 611, cover 610 and PZTtransducer 614 attached to the bottom of substrate 611. FIG. 38C showsthat after injection of entities fluid 6020 and buffer fluid 6040,entities fluid 6020 containing large entities 612 and small entities 613mainly flow along the edges of the channel 601 as laminar flow. ACvoltage is applied to PZT 614, where frequency (Fp) of AC voltage ispreferred to be at a frequency matching to the PZT resonance frequency(Fr). PZT 614 produces ultrasound vibrations to the substrate 611 atfrequency Fp. Said ultrasound vibrations transfer to the fluid containedin channel 601. Channel 601 has channel width 625 defined as the normaldistance between the two side walls of channel 601. In one embodiment,width 625 is between 100 nm to 1 um. In another embodiment, width 625 isbetween 1 um to 10 um. In yet another embodiment, width 625 is between10 um to 100 um. In yet another embodiment, width 625 is between 100 umto 500 um. In yet another embodiment, width 625 is between 500 um to 5mm. When channel width 625 is half wavelength, or an integer multiple ofhalf wavelength, of the ultrasound mode in the fluid within channel 601at frequency Fp, standing wave may be present in between the two sidewalls of channel 601 as indicated by the dashed lines 626. FIG. 38Cshows when channel width 625 is half wavelength of fluid ultrasound modeat frequency Fp, where a single fluidic pressure node 615 is formedalong the center line of channel 601 in the direction of channel length,which is perpendicular to the view of FIG. 38C. In another embodiment,channel width 625 is an integer times of half wavelength of fluidultrasound mode at frequency Fp, where integer is larger than 1, andsaid integer number of fluidic pressure nodes may then be formed acrossthe width 635 with each node being a line along the direction of channellength. Presence of standing wave 626 and pressure node 615 exertacoustic force, which is shown in FIG. 38D as arrows 628, on entities inthe entities fluid laminar flow along the side walls of channel 601 andcause large size entities 6070 to move close to center node 615 duringflowing through the channel 601. Said acoustic force 628 has thecharacteristics of: (1) largest amplitude close to channel 601 sidewalls with force direction pointing from the side walls towards the node615; (2) smallest force, or close to zero force, around node 615; (3)being linearly proportional to size of the entities; (4) is a functionof the density and compressibility of both the buffer fluid 6040 and theentities. Due to these characteristics, with proper optimization ofbuffer fluid composition, buffer fluid 6040 laminar flow speed, andentities fluid 6020 laminar flow speed, large entities 612 may beoptimized to preferably break the laminar flow barrier to enter thebuffer laminar flow due to a larger acoustic force acting on largeentities 612, and be concentrated around the center node 615.

FIG. 38D is a schematic diagram illustrating the fluid acoustic wave ofFIG. 38C causing larger size entities 612 to move into buffer fluidlaminar flow around center of the channel 601. When fluid within thechannel 601 exits the channel to outlets 607 and 609, channel 601 centersub-channel width 651 of FIG. 38A to outlet 607 may be much smaller thanthe width 625 of the channel 601, thus only allow large entities 612 atcenter flow within channel 601 to exit outlet 607 as large entities 6070fluid. While smaller entities 613 mainly in the close to side walllaminar flow exit channel 601 through side channels 308 to exit fromoutlet 609 as small entities 6090 fluid.

Frequency Fp of PZT 614 vibration in one embodiment is between 100 kHzto 500 Hz, between 500 kHz to 1 MHz in another embodiment, between 1 MHzto 3 MHz in yet another embodiment, between 3 MHz to 10 MHz in yetanother embodiment, and between 10 MHz to 100 MHz in yet anotherembodiment. In FIG. 38C and FIG. 38D, PZT 614 may also be attached totop of cover 610 in FIG. 38C and FIG. 38D, and ultrasound vibrationsfrom PZT 614 is transferred from PZT 614 through cover 610 to fluidwithin channel 601, or through cover 601 to substrate 611 and then tothe fluid within channel 601.

FIG. 39 is a schematic diagram illustrating methods to use a UFL toseparate biological entities of different sizes, where UFL may be UFL600 from FIG. 38A or FIG. 40A, UFL 620, 630 and 640 from FIG. 41Athrough FIG. 43. Sequential steps of 701 to 705 and 706 aresubstantially similar as described in FIG. 38A, FIG. 38B, FIG. 38C, andFIG. 38D, except steps 703 and 704 refer to possibility of multiplepressure nodes, as shown in FIG. 41B and FIG. 42B. Step 707 entitiesanalysis can be performed on both the large entities 6070 and smallentities 6090, and may include processes 903, 904, 905, 906, 5824, 5825,5826 as described in FIG. 53, FIG. 79, FIG. 80, FIG. 82 and FIG. 83 oncorresponding UFL output samples. Continued process 708, for examplethrough a MAG device as shown in FIG. 44A through FIG. 45C, FIG. 47through FIG. 49, or through cascaded UFL process as in FIG. 54C.

FIG. 40A is a cross-sectional view of a portion of a UFL 650 similar toFIG. 38B. UFL 650 is identical to UFL 600 from a top-down view as inFIG. 38A, except that a uniform soft magnetic layer (“SML”) 616 isdeposited on top the substrate 611 of UFL 650, and patterned togetherwith the substrate 611 to form inlets 602 and 604, outlets 607 and 609,and channel 601, 603 and 608. SML 616 may be composed of at least oneelement from iron (Fe), cobalt (Co), and nickel (Ni). SML 616 thickness6164 is between 10 nm to 100 nm in one embodiment, between 100 nm to 1um in another embodiment, between 1 um to 10 um in yet anotherembodiment, between 10 um to 100 um in yet another embodiment, between100 um to 1 mm in yet another embodiment, and between 1 mm to 3 mm inyet another embodiment. Deposition of SML layer 616 on substrate 611 maybe by any of: electro-plating, vacuum plating, plasma-vapor-deposition(PVD), atomic layer deposition (ALD), chemical vapor deposition (CVD).Etching of layer 616 together with substrate 611 to form inlets 602 and604, outlets 607 and 609, and channel 601, 603 and 608 may be by any of:dry etch, plasma enhanced dry etch, ion plasma etch, and IBE. Layer 616may be a continuous layer along the channel 601 length direction andforms as part of the side walls of the channel 601.

FIG. 40B is similar as FIG. 38D and shows a schematic diagramillustrating the large entities 612 if concentrated by acoustic force628 to the channel 601 center around the pressure node 615 and smallentities 613 mainly remain around the channel 601 side wall.Additionally, a magnetic field 617 is applied in plane and inducesmagnetization 6162 in the SML layer 616. For the SML layer 616 locatedas part of the side walls of the channel 601, magnetization 6162produces local magnetic field 6163, which has strongest magnetic fieldstrength and field gradient close to the channel 601 side walls. Field6163 may help maintain magnetic small entities, for example freemagnetic labels SPL 2 that is part of the entities fluid 6020 inpositive sample after MAG separation as shown in FIG. 82 and FIG. 83, tostay within the laminar flow close of channel 601 side walls and outputfrom outlet 609 of FIG. 38A.

FIG. 40C shows that after etching of SML layer 616 together withsubstrate 611, and before cover 610 is attached to substrate 611, apassivation layer 6172 may be deposited covering exposed surfaces of SMLlayer 616 and substrate 611, Layer 6172 may help isolate fluid reactionwith material of SML layer 616. Layer 6172 may be deposited over theetched surfaces of SML 616 and substrate 61, preferably conformably, byvacuum plating, electro-plating, PVD, ALD, CVD, molecular beamdeposition (MBE), and diamond like carbon (DLC) deposition. Layer 6172may be an oxide, or a nitride, or a carbide, of any one or more ofelements of: Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr, Zn, Mg, Nb, Mo,Ni, Co, Fe, Ir, Mn, Ru, Pd, and C. Layer 6273 may compose at least oneof: Si, Ti, Ta, Fe, Al, W, Zr, Hf, V, Cu, Cr, Zn, Mg, Nb, Mo, Ni, Co,Fe, Ir, Mn, Ru, Pd, and C. Layer 6273 may be a DLC layer. Thickness oflayer 6273 may be between 1 nm to 10 nm in one embodiment, between 10 nmto 100 nm in another embodiment, between 100 nm to 10 um in anotherembodiment, and between 10 um to 100 um in another embodiment.

FIG. 41A is a top-down view of a second UFL embodiment UFL 620, which issame as FIG. 38A, except including a wider section 6012 of the mainchannel connecting between the inlet 604, and the narrower channelsection 601 of FIG. 38A. Slope 6016 represents a transition section 6016from wider section 6012 to narrow section 601. Channel sections 6012 and601 are substantially straight and linear along channel lengthdirection. Transition section 6016 may be a section of the main channel,where the main channel includes channel section 6012 connecting throughthe transition section 6016 to channel section 601. Transition section6016 functions to funnel fluid flow from wider section 6012 into thenarrower section 601. Channel wall of transition section 6016 mayintersect with straight wall of wider section 6012 at a transition startpoint. Channel wall of transition section 6016 may intersect withstraight wall of narrower section 601 at a transition stop point. In oneembodiment, the channel shape of the transition section 6016 betweentransition start point and transition stop point may be a straight slopeas shown in FIG. 41A. In another embodiment, the channel shape of thetransition section 6016 between transition start point and transitionstop point may be a curvature, whereas the curvature may be tangentialto one of, or both of, channel wall of wider section 6012, and channelwall of narrower section 601.

FIG. 41B is a cross-sectional view of UFL 620 along direction 66 in FIG.41A, which is across the wider section 6012. Wider section 6012 has achannel width 6252, which is the full wavelength of the ultrasound modein the liquid within channel section 6012 at PZT 614 operating frequencyFp as described in FIG. 38C, and effectively twice the channel width 625of channel 601 as in FIG. 38C and FIG. 41C. Due channel width 6252 beingequal to the full wavelength of ultrasound mode at Fp, two pressurenodes may exist in channel section 6012, where acoustic force from theultrasound mode may move and concentrate large entities 612 at each ofthe two nodes from the channel wall entity laminar flow.

FIG. 41C is a cross-sectional view of UFL 620 along direction 65 in FIG.41A, which is across the narrower section 601, which is identical toFIG. 38D. After fluid within channel section 6012 flows through thetransition 6016 to channel section 601, single pressure node of channelsection 601 forces the large entities 612 to concentrate at channelsection 601 center same as in FIG. 41C. Wider section 6012 provides afirst stage large entities 612 separation from small entities 613. Aftertransition section 6016, flow rates of center buffer laminar flow andchannel side wall entities laminar flow increase to about twice thespeed of same flow in section 6012 due to the channel width reductionfrom 6252 to 625. Channel section 601 provides a second stage largeentities separation from small entities, together with the increase flowrate in channel section 601, purity of large entities 612 in 6070 fluidoutput from outlet 607, as well as purity of small entities 613 in 6090fluid output from outlet 609, may be enhanced compared to UFL 600 ofFIG. 38A.

FIG. 42A is a top-down view of a third UFL embodiment UFL 630, which isa further enhancement from the UFL 620 of FIG. 41A. Every aspect of FIG.42A is same as FIG. 41A, except that when compared to UFL 620 of FIG.41A, UFL 630 of FIG. 43A includes additional side channels 6013 thatconnect from around the transition section 6016 to side channels 608, orin another embodiment directly to the outlet 609, to divert side walllaminar flow of small entities 613 from wider section, or referred to asfirst stage section, 6012, as shown in FIG. 42B, directly to output 6090without entering narrower section, or referred to as second stagesection, 601. Channel sections 6012 and 601 are substantially straightand linear along channel length direction. In one embodiment, sidechannels 6013 connect from first stage section 6012 before thetransition start point of section 6012 intersecting section 6016. Inanother embodiment, side channels 6013 connect from the transition startpoint of section 6012 intersecting section 6016. In yet anotherembodiment, side channels 6013 connect from the within the transitionsection 6016 between the transition start point of section 6012intersecting section 6016 and the transition stop point of section 6012intersection section 601. In yet another embodiment, side channels 6013connect from the transition stop point of section 6012 intersectingsection 601. In yet another embodiment, side channels 6013 connect fromthe second stage section 601 after the transition stop point of section6012 intersecting section 601.

FIG. 42B is a cross-sectional view of UFL 630 along direction 66 in FIG.42A, which is across the wider section 6012. FIG. 42B is identical toFIG. 41B.

FIG. 42C is cross-sectional view of UFL 630 along direction 65 in FIG.42A, which is across the narrower section 601 and side channels 6013.Compared to FIG. 41C, side channels 6013 connecting from around thetransition section 6016 of FIG. 42A contains mainly, or purely, smallentities 613. While the channel 601 of FIG. 42C shows large entities 612separation and concentration to channel 601 center pressure node similaras in FIG. 41C, but small entities 613 around section 601 channel wallsis reduced in density when compared to FIG. 41C. Due to the pre-channelsection 601 small entities diversion by side channel 6013, UFL 630 mayhave an even higher purity of large entities 612 in 6070 fluid outputfrom outlet 607, as well as higher purity of small entities 613 in 6090fluid output from outlet 609.

FIG. 43 is a top-down view of a fourth UFL embodiment UFL 640 having amultiple-stage UFL channel with sequential channel width reduction alongthe channel flow path. FIG. 43 shows a further enhancement in increasinglarge entities purity in 6070 and small entities purity in 6090. FIG. 43shows an additional wider width section 6014 is added between inlet 604and channel section 6012. Channel width of 6014 may be three times ofthe half wavelength of ultrasound mode of the liquid flowing through theUFL 640 channel at PZT frequency Fp, which is one half wavelength widerthan channel width 6252 of section 6012. Channel width of 6014 may alsobe wider than the channel width 6252 of next stage channel section 6012by an integer times of the half wavelength, where said integer is largerthan one. Channel section 6014 changes to reduced channel width section6012 through a transition section 6017. Side channels 6015 connect fromaround the transition section 6017 to side channels 6013, or 608, ordirectly to outlet 609 to divert small entities 613 from channel sidewall laminar flow of section 6014 from entering section 6012, therebyincreasing purity of large entities concentration in section 6012.Channel sections 6014, 6012 and 601 are substantially straight andlinear along channel length direction.

As a further extension from FIG. 43, a multiple-stage UFL 640 may havemultiple channel sections along the UFL 640 channel flow path, whereeach earlier section of the UFL channel along the channel flow path mayhave a channel width that is wider than the immediate next sectionchannel width by an integer number of a half wavelength of ultrasoundmode in the fluid flow at the PZT frequency Fp, where said integer isequal to or larger than 1. Final channel section before flow exiting theoutlets of the UFL 640 is preferred to have a channel width equal tosaid half wavelength in one embodiment, but may also have a channelwidth that equals to an integer number of said half wavelength inanother embodiment where integer is larger than one. Side channelsconnecting to each of the transition area between adjacent channelsections divert small entities from the earlier channel in the entitieslaminar flow close to the earlier channel walls towards the outlet 609to reduce number of small entities entry into immediate next stagechannel section.

FIG. 44A through FIG. 65B illustrate various embodiments of method toutilize MAG and UFL device to separate biological entities from anentity fluid. For simplicity of description UFL 600 of FIG. 38A and MAG123 with channel 201 are used in the figures for explanation. However,UFL 600 may be replaced with UFL 650, 630, 640 of FIG. 40A, FIG. 41A,FIG. 42A, FIG. 43, while MAG 123 may be replaced with MAG 121, 122, 124,124,125, 126, 127, 128, 129 and corresponding channel types as describedin prior figures without limitation and without sacrifice ofperformance.

FIG. 44A illustrates first type sample processing method wherebiological sample is first passed through UFL 600 and the large entityoutput 6070 of UFL 600 is then passed through channel 201 adapted to MAG123, with a first type flow connector 801 connecting the UFL 600 largeentity outlet 607 and MAG inlet flow as in step 401 of FIG. 31 or step708 of FIG. 39. For the in series operation of UFL 600 and MAG 123devices, optimal flow rate for UFL channel 601 and optimal flow rate forMAG channel 201 may be different. Optimal flow rate for UFL channel 601acoustic force separation of large and small entities are determined bylaminar flow condition, and separation efficiency between large andsmall entities. Optimal flow rate for MAG 123 separation is determinedby the length of channel 201 and magnetic field force on magnetic labelsattached to the entities. Direct fluidically coupled flow from UFL 600outlet 607 to MAG channel 201 inlet will force the flow rate being thesame through UFL 600 channel and MAG channel 201, which may incurnegative impact on separation efficiency for either one or both of UFL600 and MAG 123. It is necessary to decouple the fluid flows through UFL600 and MAG 123 channel 201. Flow connector 801 serves to decouple flowrates of the UFL 600 and MAG 123. Output fluid 6070 is first injectedinto connector 801 through inlet 8011, and fluid in connector 801 isoutput through outlet 8012 as flow as in steps 401/708 into inlet ofchannel 201 of MAG 123. Both UFL 600 and MAG 123 channel 201 may operateat their respective optimal flow rate. In one embodiment where MAG 123channel 201 optimal flow rate is larger than UFL 600 optimal flow rate,MAG 123 extracts fluid 401/708 from connector 801 faster than UFL 600injects fluid 6070 into the connector 801. A fluid level sensor 100 maybe attached to connector 801 to sense fluid level remaining in connector801. If fluid level drops below a low threshold, sensor 100 may signalMAG 123 to pause flow as in steps 401/708 intake to wait for connector801 internal liquid level to increase to another higher level before MAG123 may restart extracting fluid as in steps 401/708 from connector 801.In another embodiment where MAG 123 channel 201 optimal flow rate issmaller than UFL 600 optimal flow rate, MAG 123 extracts fluid as insteps 401/708 from connector 801 slower than UFL 600 injects fluid 6070into the connector 801. If fluid level increases above a low threshold,sensor 100 may signal UFL 600 to pause flow 6070 output to wait forconnector 801 internal liquid level to drop to another lower levelbefore UFL 600 may restart outputting fluid 6070 into connector 801.Flow connector 801 may be in the design as shown in FIG. 44A, whereinlet 8011 is at a higher vertical location than outlet 8012, where flow6070 enters connector 801 and accumulates at outlet 8012 at inside of801 due to gravity. Alternatively, liquid sample may be completelyprocessed through UFL 600 first and stored in connector 801. MAG 123then extracts fluid from connector 801 as input into the MAG 123 channel201 and completes processing of all liquid sample from connector 801.Connector 801 may be made as part of an enclosed fluidic line, whereduring the path of flow 6070 from UFL 600 outlet 607 to inlet 8011 ofconnector 801, to outlet 8012, to flow as in steps 401/708 into inlet ofchannel 201, fluid sample is not exposed to air, and being sterile.

FIG. 44B illustrates first type sample processing method of FIG. 44Awith using a second type flow connector 802 connecting the UFL 600 largeentity outlet 607 and MAG 123 channel 201 inlet. Connector 802 as shownin FIG. 44B takes the form similar to a vial. Flow 6070 enters connector802 through a short length inlet tube 8021 of connector 802 and drips tobottom of the connector 802 due to gravity. Flow as in steps 401/708 isextracted from the fluid at the bottom of the connector 802 by a longlength outlet tube 8022 to input of channel 201. Fluid level sensor 100may be attached to connector 802 to sense fluid level within connector802. UFL 600 and MAG 123 may both operate at their respective optimalflow rate, and fluid level sensor 100 may function to pause UFL 600operation or MAG 123 operation with the same method as described in FIG.44A. Alternatively, liquid sample may be completely processed throughUFL 600 and stored in connector 802. MAG 123 then extracts fluid fromconnector 801 as input into the MAG 123 channel 201 and completesprocessing of all liquid sample from connector 802. Connector 802 may bemade as part of an enclosed fluidic line similar as connector 801.

FIG. 44C illustrates first type sample processing method of FIG. 44Awith using a third type flow connector 803 connecting the UFL 600 largeentity outlet 607 and MAG 123 channel 201 inlet. Connector 803 as shownin FIG. 44C takes the form similar to a fluid bag or blood bag. Flow6070 enters connector 803 through a bottom inlet 8031 fills connector803 from bottom of the connector 803 due to gravity. Flow as in steps401/708 is extracted from the fluid at the bottom of the connector 803through outlet 8032 to input of channel 201. Fluid level sensor 100 maybe attached to connector 803 to sense fluid level within connector 803.UFL 600 and MAG 123 may both operate at their respective optimal flowrate, and fluid level sensor 100 may function to pause UFL 600 operationor MAG 123 operation with the same method as described in FIG. 44A.Alternatively, liquid sample may be completely processed through UFL 600and stored in connector 803. MAG 123 then extracts fluid from connector801 as input into the MAG 123 channel 201 and completes processing ofall liquid sample from connector 803. Connector 803 may be made as partof an enclosed fluidic line similar as connector 801.

FIG. 45A illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with first typeflow connector 801 connecting the UFL small entity 6090 outlet 609 andMAG 123 channel 201 inlet. FIG. 45A is identical to FIG. 44A in everyaspect except small entities flow 6090 from outlet 609 is injected intothe inlet 8011 of connector 801.

FIG. 45B illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with second typeflow connector 802 connecting the UFL small entity 6090 outlet 609 andMAG 123 channel 201 inlet. FIG. 45B is identical to FIG. 44B in everyaspect except small entities flow 6090 from outlet 609 is injected intothe inlet 8021 of connector 802.

FIG. 45C illustrates second type sample processing method wherebiological sample is first passed through UFL 600 and the small entityoutput 6090 of UFL 600 is then passed through MAG 123, with third typeflow connector 803 connecting the UFL small entity 6090 outlet 609 andMAG 123 channel 201 inlet. FIG. 45C is identical to FIG. 44C in everyaspect except small entities flow 6090 from outlet 609 is injected intothe inlet 8031 of connector 803.

FIG. 46A illustrates third type sample processing method wherebiological sample is first passed through MAG 123 channel 201, andfollowing procedure 427 or 428 of FIG. 31, the output of MAG 123 channel201 is then passed through UFL 600 as entity fluid 6020 into inlet 602as in step 408 of FIG. 31, with first type flow connector 801 connectingthe MAG 123 channel 201 outlet and UFL 600 entity fluid 6020 inlet 602.In FIG. 46A, output from MAG 123 can be either negative entities that donot have attached SPL 2, or positive entities separated by MAG 123magnetic field and subsequently dissociated and flushed out of channel201 as described in FIG. 31. Similar as in FIG. 44A, MAG 123 and UFL 600may each operate with their respective optimal flow rate. Fluid levelsensor 100 may be attached to connector 801 to sense fluid levelremaining in connector 801. Fluid level sensor 100 operates similarly asin FIG. 44A to sense fluid in connector 801, and depending on the flowrate difference between MAG 123 and UFL 600, may pause MAG 123 or UFL600 flow to maintain fluid level in connector 801 above a low level orbelow a high level. Alternatively, liquid sample may be completelyprocessed through MAG 123 first and stored in connector 801. UFL 600then extracts fluid from connector 801 as input into the inlet 602 andcompletes processing of all liquid sample from connector 801. Connector801 may be made as part of an enclosed fluidic line similarly as in FIG.44A.

FIG. 46B is same as FIG. 46A in every aspect, except replacing connector801 with connector 802, where operation of connector 802 and attachedsensor 100 is same as described FIG. 44B.

FIG. 46C is same as FIG. 46A in every aspect, except replacing connector801 with connector 803, where operation of connector 803 and attachedsensor 100 is same as described FIG. 44C.

FIG. 47 illustrates fourth type sample processing method wherebiological sample is first passed through multiple UFLs 600, outputfluids from the UFLs 600, which can be either large entities 6070 orsmall entities 6090, are then fed into inlets 8011 of a fourth type flowconnector 8010, and from connector 8010 outlets 8012 into the inlets ofchannels 201 of multiple MAGs 123. FIG. 47 is functionally similar toFIG. 44A and FIG. 45A. Connector 8010 is also functionally same asconnector 801, except inlet 8011 of connector 8010 accept multiple fluidoutput from multiple UFLs 600, and outlet 8012 of connector 8010 outputsto input of multiple channels 201 of multiple MAGs 123.

FIG. 48 illustrates fifth type sample processing method where biologicalsample is first passed through multiple UFLs 600, output fluids from theUFLs 600, which can be either large entities 6070 or small entities6090, are then fed into inlets 8021 of a fifth type flow connector 8020,and from connector 8020 outlets 8022 into the inlets of channels 201 ofmultiple MAGs 123. FIG. 48 is functionally similar to FIG. 44B and FIG.45B. Connector 8020 is also functionally same as connector 802, exceptinlet 8021 of connector 8020 accept multiple fluid output from multipleUFLs 600, and outlet 8022 of connector 8020 outputs to input of multiplechannels 201 of multiple MAGs 123.

FIG. 49 illustrates sixth type sample processing method where biologicalsample is first passed through multiple UFLs 600, output fluids from theUFLs 600, which can be either large entities 6070 or small entities6090, are then fed into inlets 8031 of a sixth type flow connector 8030,and from connector 8030 outlets 8032 into the inlets of channels 201 ofmultiple MAGs 123. FIG. 49 is functionally similar to FIG. 44C and FIG.45C. Connector 8030 is also functionally same as connector 803, exceptinlet 8031 of connector 8030 accept multiple fluid output from multipleUFLs 600, and outlet 8032 of connector 8030 outputs to input of multiplechannels 201 of multiple MAGs 123.

In each of FIG. 47, FIG. 48, and FIG. 49, in one embodiment, samebiological sample is divided and processed simultaneously throughmultiple UFLs 600. In another embodiment, each of the UFLs 600 processesa different biological sample. Output from each UFL 600, either largeentities 6070 fluid from outlet 607 or small entities fluid from outlet609, shown as dashed lines in FIG. 47, FIG. 48 and FIG. 49, may beindividually input into the inlet 8011 of connector 8010 of FIG. 47, orinto inlet 8021 of connector 8020 of FIG. 48, or into inlet 8031 ofconnector 8030 of FIG. 49, as shown by solid lines 6070/6090 in each ofFIG. 47, FIG. 48 and FIG. 49. From outlet 8012, 8022, 8032 of FIG. 47,FIG. 48 and FIG. 49 respectively, following steps of 401 or 708, each ofthe MAGs 123 of FIG. 47, FIG. 48, or FIG. 49 may extract fluid samplefrom corresponding connector 8010, 8020, and 8030 into its correspondingchannel 201. Each of the UFL 600 and each of the MAG 123 of FIG. 47,FIG. 48, or FIG. 49 may operate at its own respective optimal sampleflow rate, which may be different between different UFLs 600 anddifferent between different MAGs 123 within same figure. Due to theexistence of the connector 8010, 8020, and 8030, flow rate interferencebetween the different UFLs 600 and MAGs 123 within each of FIG. 47, FIG.48 and FIG. 49 are minimized or eliminated. Fluid level sensor 100 maybe attached to buffers 8010, 8020, and 8030 to sense fluid levelremaining in each of the flow connectors 8010, 8020, and 8030. Fluidlevel sensor 100 operates similarly as in FIG. 44A through FIG. 44C insensing fluid in flow connectors 8010, 8020, and 8030, and depending onthe flow rate difference between MAGs 123 and UFLs 600 of each figure,may pause operation of one or more MAGs 123, or may pause operation ofone or more UFLs 600 of each figure to maintain fluid level incorresponding connector 8010, 8020, or 8030 to be above a low levelthreshold or below a high level threshold. Alternatively, liquid samplemay be completely processed through all UFLs 600 first and stored incorresponding connector 8010, 8020, or 8030 of each FIG. 47, FIG. 48 andFIG. 49. MAGs 123 then extract fluid from corresponding connector 8010,8020, or 8030 of each figure and complete processing of all liquidsample from each corresponding connector 8010, 8020, or 8030. Connectors8010, 8020, and 8030 may each be made as part of a set of enclosedfluidic lines, which may include UFLs 600, channels 201 and connectionsfrom UFLs 600 to each connector 8010, 8020, 8030 and from each connector8010, 8020, and 8030 to channels 201, similar as described in FIG. 44Athrough FIG. 44C.

FIG. 50 illustrates seventh type sample processing method wherebiological sample is first passed through multiple MAGs 123, outputfluids from the MAGs 123 channels 201 are then fed into inlets 8011 offlow connector 8010 of FIG. 47, and from flow connector 8010 outlets8012 into the entity fluid inlets 602 of multiple UFLs 600.

FIG. 51 illustrates eighth type sample processing method wherebiological sample is first passed through multiple MAGs 123, outputfluids from the MAGs 123 channels 201 are then fed into inlets 8021 offlow connector 8020 of FIG. 48, and from flow connector 8020 outlets8022 into the entity fluid inlets 602 of multiple UFLs 600.

FIG. 52 illustrates ninth type sample processing method where biologicalsample is first passed through multiple MAGs 123, output fluids from theMAGs 123 channels 201 are then fed into inlets 8031 of flow connector8030 of FIG. 49, and from flow connector 8030 outlets 8032 into theentity fluid inlets 602 of multiple UFLs 600.

In each of FIG. 50, FIG. 51, and FIG. 52, in one embodiment, samebiological sample is divided and processed simultaneously throughmultiple MAGs 123. In another embodiment, each of the MAGs 123 processesa different biological sample. Output from each MAG 123, either negativeentities following procedure 427, or positive entities followingprocedure 428, may be individually input into the inlet 8011 ofconnector 8010 of FIG. 50, or into inlet 8021 of connector 8020 of FIG.51, or into inlet 8031 of connector 8030 of FIG. 52, as shown by solidlines 427/428 in each of FIG. 50, FIG. 51 and FIG. 52. From outlet 8012,8022, 8032 of FIG. 50, FIG. 51 and FIG. 52 respectively, following stepof 408, each of the UFLs 600 of FIG. 50, FIG. 51, or FIG. 52 may extractfluid sample as entity fluid 6020 from corresponding connector 8010,8020, and 8030 into its corresponding entities inlet 602, Each of theUFL 600 and each of the MAG 123 of FIG. 50, FIG. 51, or FIG. 52 mayoperate at its own respective optimal sample flow rate, which may bedifferent between different UFLs 600 and different between differentMAGs 123 within same figure. Due to the existence of the connector 8010,8020, and 8030, flow rate interference between the different UFLs 600and MAGs 123 within each of FIG. 50, FIG. 51 and FIG. 52 are minimizedor eliminated. Fluid level sensor 100 may be attached to flow connectors8010, 8020, and 8030 to sense fluid level remaining in each of the flowconnectors. Fluid level sensor 100 operates similarly as in FIG. 46Athrough FIG. 47C in sensing fluid in flow connectors 8010, 8020, and8030, and depending on the flow rate difference between MAGs 123 andUFLs 600 of each figure, may pause operation of one or more MAGs 123, ormay pause operation of one or more UFLs 600 of each figure to maintainfluid level in corresponding connector 8010, 8020, or 8030 to be above alow level threshold or below a high level threshold. Alternatively,liquid sample may be completely processed through all MAGs 123 first andstored in corresponding connector 8010, 8020, or 8030 of each FIG. 50,FIG. 51 and FIG. 52. UFLs 600 then extract fluid from correspondingconnector 8010, 8020, or 8030 of each figure and complete processing ofall liquid sample from each corresponding connector 8010, 8020, or 8030.Flow connectors 8010, 8020, and 8030 may each be made as part of a setof enclosed fluidic lines, which may include UFLs 600, channels 201 andconnections from channels 201 to each connector 8010, 8020, 8030 andfrom each connector 8010, 8020, and 8030 to UFLs 600, similar asdescribed in FIG. 46A through FIG. 46C.

FIG. 53 illustrates tenth type sample processing method where biologicalsample after being passed through one or more of UFLs 600 or MAGs 123,output fluids from the UFLs 600 and MAGs 123 are fed into inlets of aflower connector 8020 or a flow connector 8030, and from the flowconnectors 8020 and 8030 outlets into different type of cell processingdevices. FIG. 53 shows example of liquid sample output from MAG 123channel 201, including negative entities following procedure 427 andpositive entities following procedure 428, may be injected to inlet 8021of connector 8020 or inlet 8031 of connector 8030 similar as in FIG. 51and FIG. 52. Alternatively, UFL 600 large entities output 6070 fromoutlet 607 or small entities output 6090 from outlet 609 may be alsoinjected into to inlet 8021 of connector 8020 or inlet 8031 of connector8030 similar as in FIG. 48 and FIG. 49. After sample fluid is completelyprocessed through UFL 600 or MAG 123, and injected into, and storedwithin, connector 8020 or connector 8030, entities analysis as in step407 of FIG. 31 and step 707 of FIG. 39 may be performed by sendingsample fluid containing entities from connector 8020 or connector 8030to any of: cell counter 903, cell imager 904, flow cytometer or sorter905, and DNA or RNA sequencer 906. Entities may be further sent to DNAor RNA sequencer 906 after cell counter 903 as in 936, or after cellimager 904 as in 946, or after flow cytometer or sorter 905 as in 956.For sending the sample fluid from outlet 8022 of connector 8020, or fromoutlet 8032 of connector 8030, pressurized chamber 800 may be used tocontain the connector 8020 or connector 8030 inside, and force samplefluid out of connector 8020 or connector 8030 in a steady and continuousflow stream. Chamber 800 may be a chamber filled with pressurized airinside. Connector 8020 in vial type may have an additional air port 8023open to chamber 800 internal pressurized air to help push sample fluidout of connector 8020. While connector 8030 may be in a flexible bloodbag form, which when under pressured air of chamber 800, willautomatically deflate and force sample liquid out through outlet 8032.To avoid back flow into UFL 600 or MAG 123 channel 201, shut off valves805 may be implemented on output lines from MAG 123 channel 201 and UFL600 to connector 8020 or connector 8030.

FIG. 54A illustrates eleventh type sample processing method wherebiological sample after being passed through a first MAG 123 channel 201during a magnetic separation may output negative entities fluidfollowing procedure 427, or positive entities fluid following procedure428, into inlet of a second MAG 123 channel 201 input as in step 408 ofa continued process. FIG. 54A illustrates a multi-stage MAG process.

FIG. 54B illustrates twelfth type sample processing method where afterbiological sample passed through MAG 123 for magnetic separation, outputfluid from MAG 123 channel 201, containing either negative entities orposition entities, may be diverted through a T-connector 912 into flow913. Flow 913 may then be re-input back into the input of the channel201 of MAG 123 for another round of magnetic separation throughT-connector 911. T-connector 911 allows initial fluid sample input as instep 401 and recycled flow 913 input to channel 201. T-connector 912allows output from channel 201 into recycled flow 913 or output from MAG123 as in procedures 427 and 428. In one embodiment, recycled flow 913contains negative entities, repeated magnetic separation in FIG. 54Bhelps achieve complete depletion of all magnetic entities in thenegative entities flow before output into 427/428 procedure. In anotherembodiment, recycled flow 913 contains positive entities afterdissociation, repeated process as in FIG. 54B helps increase purity inpositive magnetic entities to allow wash off of non-magnetic entitiesthat may be in the conglomerate by non-specific bindings. FIG. 54Billustrates using same MAG 123 as a multi-cycle MAG process.

FIG. 54C illustrates thirteenth type sample processing method wherebiological sample after being passed through a first UFL 600, outputfluids from first UFL 600, for example large entities 6070 fluid fromoutlet 607 or small entities 6090 fluid from outlet 609, may be passedinto entity fluid inlets 602 of one or more subsequent UFLs 600 as amulti-stage UFL process.

FIG. 55A illustrates first embodiment of closed and disposable fluidiclines for third type sample processing method as shown in FIG. 46A,where connector 801 may be replaced with connector 802 or connector 803without limitation. Input line 923 may connect to a sample liquidcontainer. Input line 924 may connect to a MAG buffer container. Inputline 923 and input line 924 are connected through a T-connector 921 tothe inlet of the first pump tubing 504/505 that may be mounted into aperistaltic pump. First pump tubing 504/505 outlet connects to channel201 which may be used as part of MAG 123. Output of channel 201 connectsto T-connector 922, which connects to output line 925 and output line926. Output line 925 may connect to an MAG out sample container andoutput line 926 connects to inlet of connector 801. In one embodiment,output line 925 may output negative entities to said MAG out samplecontainer, and output line 926 may output positive entities to connector801. In another embodiment, output line 925 may output positive entitiesto said MAG out sample container, and output line 926 may outputnegative entities to connector 801. Connector 801 outlet connects toinput line 9271 of a second pump tubing 504/505. Said second pump tubing504/505 outlet then connects to UFL 600 sample input line 6020. Inputline 9272 may connect to UFL buffer container and connects to inlet of athird pump tubing 504/505. Said third pump tubing 504/505 outlet thenconnects to UFL 600 buffer input line 6040. UFL 600 large entities 6070output line may connect to a large entities sample container. UFL 600small entities 6090 output line may connect to a small entities samplecontainer. FIG. 55A illustrates that besides the input and output lines923, 924, 925, 9272, 6070, and 6090 that connect to external containers,entire fluid path from sample liquid input to line 923, to sample outputto lines 925, 6070, 6090, all pumps, MAG 123 and other fluidic linecomponents will be externally attached to the lines of FIG. 55A. Thus,lines of FIG. 55A are internally enclosed, suitable for single usedisposable purpose and sterile applications.

FIG. 55B illustrates fluidic lines of FIG. 55A being connected to, orattached with, various fluidic components. Input line 923 connects to aliquid sample container 928 in blood bag form. Input line 924 connectsto buffer container 929. Valves 935 and 936 are attached to lines 923and 924 to control either sample liquid from bag 928 or buffer fromcontainer 929 is flown through T-connector 921 into first pump tubing504/505. First, second and third pump tubing 504/505 is each installedinto a peristaltic pump 500. Three pumps 500 operate to pump eithersample fluid or buffer fluid into MAG 123 and UFL 600. A flow limiter509/510 may be attached to the output line from each pump 500, includinglines 201, 6020, 6040 to reduce flow rate pulsation from the pumps 500.Channel line 201 is mounted into MAG 123. Output line 925 connects toMAG out sample container 934. Valve 940 is attached to line 925 andvalve 937 is attached to line 926, which control negative entities orpositive entities from MAG 123 going into either container 934 or theconnector 801 through the T-connector 922. Valves 940 and 937 may bothshut down the flow in lines 925 and 926 duringdemagnetization/dissociation process of MAG 123. Input line 9272 mayconnect to UFL buffer container 931. UFL output line 6070 connects tolarge entities container 932 and output line 6090 connects to smallentities 933. Adjustable valves 939 and 938 may be attached to the lines6070 and 6090 to adjust the flow rate within each line of 6070 and 6090,which in turn controls the laminar flow speed in UFL channel for channelcenter buffer flow and channel edge entities sample flow.

FIG. 56A illustrates second embodiment of closed and disposable fluidiclines for third type sample processing method as shown in FIG. 46A. FIG.56A is identical to FIG. 55A, except the output line 925 is connected toa MAG sample container 934, UFL output line 6070 is connected to a largeentities container 932, and UFL output line 6090 is attached to a smallentities container 933. FIG. 56A illustrates containers 934, 932, 933are in the form of blood bags. Bags 932, 933, 934 as part of the encloselines of FIG. 56A may be disposable and made sterile, and may also beseparated from the lines after separation process for steps 407 and 408of FIG. 31, or steps 707 and 708 of FIG. 39.

FIG. 56B describes the identical process of: connecting sample container928, buffer container 929, buffer container 931 to the lines 923, 924and 9272 respectively, same as in FIG. 55B. Containers 928, 929, 931 arein blood bag form. Also same as described in FIG. 55B, three pump tubing504/505 are installed in the three peristaltic pumps 500, valves 935,936, 940, 937, 939, 938, are each attached to the corresponding lines,and flow limiters 509/510 may be attached at output line of each pump500 same as in FIG. 55B.

FIG. 57A illustrates embodiment of closed and disposable fluidic linesfor first type sample processing method as shown in FIG. 44A, whereconnector 801 may be replaced with connector 802 or connector 803without limitation. Input line 9271 may connect to a UFL sample liquidcontainer, and also connects to inlet of a first pump tubing 504/505,which further connect to entities input line 6020 of UFL 600. Input line9272 may connect to a UFL buffer container, and also connects to inletof a second pump tubing 504/505, which further connect to buffer inputline 6040 of UFL 600. UFL 600 large entities output line 6070 connectsto inlet of connector 801. UFL 600 small entities output line 6090 mayconnect to a small entities container. Outlet of connector 801 connectsto MAG sample input line 923. MAG buffer input line 924 may connect to aMAG buffer container. Input lines 923 and 924 are connected through aT-connector 921 to the inlet of the third pump tubing 504/505. Thirdpump tubing 504/505 outlet connects to channel 201 which may be used aspart of MAG 123. Output of channel 201 connects to T-connector 922,which connects to output line 925 and output line 926. Output lines 925and 926 may each connect to an MAG out sample container. In oneembodiment, output line 925 may output negative entities to a first MAGout sample container, and output line 926 may output positive entitiesto a second MAG out sample container. FIG. 57A illustrates that besidesthe input and output lines 9271, 9272, 924, 6090, 925, and 926 thatconnect to external containers, entire fluid path from UFL sample andUFL buffer input lines 9271 and 9272, to sample output lines 6090, 925and 926, all pumps, MAG 123 and other fluidic line components will beexternally attached to the lines of FIG. 57A. Thus, lines of FIG. 57Aare internally enclosed, suitable for single use disposable purpose andsterile applications.

FIG. 57B illustrates fluidic lines of FIG. 57A being connected to, orattached with, various fluidic components. First, second and third pumptubing 504/505 is each installed into a peristaltic pump 500. Threepumps 500 operate to pump either sample fluid or buffer fluid into MAG123 and UFL 600. A flow limiter 509/510 may be attached to the outputline from each pump 500, including lines 201, 6020, 6040 to reduce flowrate pulsation from the pumps 500. Input line 9271 connects to a liquidsample container 928 in blood bag form. Input line 9272 connects to UFLbuffer container 931 also in blood bag form. UFL output line 6090connects to small entities container 933 in blood bag form. Adjustablevalves 939 and 938 may be attached to the lines 6070 and 6090 to adjustthe flow rate within each line of 6070 and 6090, which in turn controlsthe laminar flow speed in UFL 600 channel for channel center buffer flowand channel edge entities sample flow. Input line 924 connects to MAGbuffer container 929. Valves 935 and 936 are attached to lines 923 and924 to control either sample liquid from connector 801 or buffer fluidfrom container 929 is flown through T-connector 921 into third pumptubing 504/505. Channel line 201 is mounted into MAG 123. Output line925 connects to first MAG out sample container 934. Output line 926connects to second MAG out sample container 9342. Valve 940 is attachedto line 925 and valve 937 is attached to line 926, which controlnegative entities and positive entities from MAG 123 going into eithercontainer 934 or container 9342 through the T-connector 922. Valves 940and 937 may both shut down the flow in lines 925 and 926 duringdemagnetization/dissociation process of MAG 123.

FIG. 58A illustrates embodiment of closed and disposable fluidic linesfor second type sample processing method as shown in FIG. 45A. FIG. 58Ais identical to FIG. 57A in every aspect, except the UFL 600 smallentities output line 6090 connects to the inlet of the connector 801instead of the output line 6070 as in FIG. 57A. Large entities outputline 6070 of FIG. 58A may connect to a large entities container.

FIG. 58B illustrates fluidic lines of FIG. 58A being connected to, orattached with, various fluidic components. FIG. 58B is identical to FIG.57B in every aspect, except the UFL 600 small entities output line 6090connects to the inlet of the connector 801 instead of the output line6070 as in FIG. 57B. Large entities output line 6070 of FIG. 58Bconnects to a large entities container 932 in blood bag form.

FIG. 59A illustrates embodiment of closed and disposable fluidic linesfor sample processing through a single MAG. Input line 923 may connectto a sample liquid container. Input line 924 may connect to a MAG buffercontainer. Input line 923 and input line 924 are connected through aT-connector 921 to the inlet of the pump tubing 504/505 that may bemounted into a peristaltic pump. Pump tubing 504/505 outlet connects tochannel 201 which may be used as part of MAG 123. Output of channel 201connects to T-connector 922, which connects to output line 925 andoutput line 926. Output lines 925 and 926 may each connect to an MAG outsample container.

FIG. 59B illustrates fluidic lines of FIG. 59A being connected to, orattached with, various fluidic components. Input line 923 connects to aliquid sample container 928. Input line 924 connects to buffer container929. Valves 935 and 936 are attached to lines 923 and 924 to controleither sample liquid from bag 928 or buffer from container 929 is flownthrough T-connector 921 into first tubing 504/505. Pump tubing 504/505is installed into a peristaltic pump 500. Pump 500 operates to pumpeither sample fluid or buffer fluid into MAG 123. A flow limiter 509/510may be attached to the output line 201 from pump 500 to reduce flow ratepulsation from the pumps 500. Channel line 201 is mounted into MAG 123.Output line 925 connects to MAG out sample container 934. Output line926 connects to MAG out sample container 9342. Valve 940 is attached toline 925 and valve 937 is attached to line 926, which control negativeentities and positive entities from MAG 123 going into either container934 or container 9342. Valves 940 and 937 may both shut down the flow inlines 925 and 926 during demagnetization/dissociation process of MAG123. FIG. 59B shows containers 928, 929, 934 and 9342 may be in the formof blood bags, but may also be in other physical forms of vial orbottles.

FIG. 60A illustrates embodiment of closed and disposable fluidic linesfor sample processing through a single UFL 600. Input line 9271 mayconnect to a UFL sample liquid container, and also connects to inlet ofa first pump tubing 504/505, which further connects to entities inputline 6020 of UFL 600. Input line 9272 may connect to a UFL buffercontainer, and also connects to inlet of a second pump tubing 504/505,which further connects to buffer input line 6040 of UFL 600. UFL 600large entities output line 6070 may connect to a large entitiescontainer. UFL 600 small entities output line 6090 may connect to asmall entities container.

FIG. 60B illustrates fluidic lines of FIG. 60A being connected to, orattached with, various fluidic components. First and second pump tubing504/505 is each installed into a peristaltic pump 500. The two pumps 500operate to pump sample fluid and buffer fluid into UFL 600. A flowlimiter 509/510 may be attached to the output line from each pump 500,including lines 6020 and 6040 to reduce flow rate pulsation from thepumps 500. Input line 9271 connects to a liquid sample container 928.Input line 9272 connects to UFL buffer container 931. UFL output line6070 connects to large entities container 932. UFL output line 6090connects to small entities container 933. Adjustable valves 939 and 938may be attached to the lines 6070 and 6090 to adjust the flow ratewithin each line of 6070 and 6090, which in turn controls the laminarflow speed in UFL 600 channel for channel center buffer flow and channeledge entities sample flow. FIG. 60B shows containers 928, 931, 932 and933 may be in the form of blood bags, but may also be in other physicalforms of vial or bottles without limitation.

FIG. 61A illustrates replacing peristaltic pumps of FIG. 56B with usingpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. In FIG. 61A, pumps 500, pump tubing 504/505, and flowlimiters 509/510 of FIG. 56B are removed. Channel 201 is connecteddirectly to T-connector 921. Connector 801 is replaced with connector803 bag. UFL entity liquid line 6020 is connected to connector 803.Sample liquid bag 928, MAG buffer bag 929, connector 803 bag and UFLbuffer bag 931 are each enclosed in a pressure chamber 800. Pressurechamber 800 may operate by increasing pressure of chamber medium, forexample air or other fluid, where the bags enclosed in chambers aresubmerged in the chamber medium. With increase of chamber mediumpressure, liquid contained in the bags may be forced out of the bags andinto the fluid lines. FIG. 61A operation may need separate MAG 123 andUFL 600 operations. At first stage, valve 941 attached to line 6020closes. Pressure in chambers 800 enclosing bags 803 and 931 is released.Pressure in chambers 800 enclosing bags 928 and 929 are increased toforce sample fluid or buffer fluid into channel 201 to start MAG 123separation. After MAG 123 separation and sample fluid in bag 928 isdepleted, bag 934 and connector 803 are each filled with output samplesfrom MAG 123 after MAG separation. Then, at second stage, valve 937 isclosed and valve 941 is open. Chambers 800 around connector 803 and bag931 increase in pressure to force connector 803 sample and buffer fluidin 931 to flow into the UFL 600 to start UFL separation. After sample inconnector 803 is depleted, and UFL 600 separation finish, bags 932 and933 contain large and small entities from UFL output. Connector 803maybe replaced by connector 8020 of FIG. 52 which has an air port 8023.

FIG. 61B illustrates replacing peristaltic pumps of FIG. 56B with usingvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 61B is same as FIG. 61A, except pressure chambers 800 areremoved. Bag 934, 932, 933, and connector 803 are each enclosed in avacuum chamber 806. Vacuum chamber 806 may operate by increasing vacuumlevel within each chamber 806, where fluid from the fluid linesconnected to the bags is forced into the bags enclosed in chambers dueto fluid line pressure being larger than the vacuum pressure. FIG. 61Boperation may also need separate MAG 123 and UFL 600 operations. Atfirst stage, valve 941 attached to line 6020 closes. Vacuum in chambers806 enclosing bags 932 and 933 is released. Vacuum in chambers 806enclosing bags 934 and 803 are increased to force sample fluid or bufferfluid into channel 201 to start MAG 123 separation. After MAG 123separation and sample fluid in bag 928 is depleted, bag 934 andconnector 803 are each filled with output samples from MAG 123 after MAGseparation. Then, at second stage, valve 937 is closed and valve 941 isopen. Vacuum in chamber 806 around connector 803 is released. Vacuum inchambers 806 enclosing bags 932 and 933 are increased to force connector803 sample and buffer fluid in 931 to flow into the UFL 600 to start UFLseparation. After sample in connector 803 is depleted, and UFL 600separation finish, bags 932 and 933 contain large and small entitiesfrom UFL output. Connector 803 maybe replaced by connector 8020 of FIG.52 which has an air port 8023.

FIG. 62A illustrates replacing peristaltic pumps of FIG. 57B with usingpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. In FIG. 62A, pumps 500, pump tubing 504/505, and flowlimiters 509/510 of FIG. 57B are removed. Channel 201 is connecteddirectly to T-connector 921. Connector 801 is replaced with connector803 bag. MAG sample line 923 is connected to connector 803. Sampleliquid bag 928, MAG buffer bag 929, connector 803 bag and UFL buffer bag931 are each enclosed in a pressure chamber 800. FIG. 62A may separateUFL 600 and MAG 123 operations. At first stage, valve 935 attached toline 923 closes. Pressure in chambers 800 enclosing bags 803 isreleased. Pressure in chambers 800 enclosing bags 928 and 931 areincreased to force sample fluid and UFL buffer fluid into UFL 600 inletsto start UFL 600 separation. After UFL 600 separation and sample fluidin bag 928 is depleted, bag 933 contains small entities fluid andconnector 803 contains large entities fluid from UFL 600 separation.Then, at second stage, valve 939 is closed and valve 935 is open.Chambers 800 around connector 803 and bag 929 increase in pressure toforce connector 803 large entities fluid sample or MAG buffer fluid in929 to flow into channel 201 of MAG 123 to start MAG 123 separation.After sample in connector 803 is depleted, and MAG 123 separationfinish, bags 934 and 9342 contain positive sample and negative samplefrom MAG 123 channel 201 output. Connector 803 maybe replaced byconnector 8020 of FIG. 52.

FIG. 62B illustrates replacing peristaltic pumps of FIG. 57B with usingvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 62B is same as FIG. 62A, except pressure chambers 800 areremoved. Bag 934, 9342, 933, and connector 803 are each enclosed in avacuum chamber 806. FIG. 62B operation may separate MAG 123 and UFL 600operations. At first stage, valve 935 attached to line 923 closes.Vacuum in chambers 806 enclosing bags 933 and 803 are increased to forcesample fluid and UFL buffer fluid into inlets of UFL 600 to start UFL600 separation. After UFL 600 separation and sample fluid in bag 928 isdepleted, bag 933 contains small entities fluid and connector 803contains large entities fluid from UFL 600 separation. Then, at secondstage, valves 938 and 939 are closed and valve 923 is open. Vacuum inchamber 806 around connector 803 is released. Vacuum in chambers 806enclosing bags 934 and 9342 are increased to force connector 803 largeentities sample or MAG buffer fluid in 929 to flow into channel 201 ofMAG 123 to start MAG 123 separation. After sample in connector 803 isdepleted, and MAG 123 separation finish, bags 934 and 9342 containpositive sample and negative sample from MAG 123 channel 201 output.Connector 803 maybe replaced by connector 8020 of FIG. 52.

FIG. 63A illustrates replacing peristaltic pumps of FIG. 58B with usingpressurized chambers 800 on input sample bags to drive fluid throughfluidic lines. FIG. 63A is identical to FIG. 62A in fluid line layoutand in operation of UFL 600 and MAG 123 with chambers 800, except thatthe UFL large entities output 6070 connects to large entities container932 in blood bag form, and small entities output 6090 connects toconnector 803.

FIG. 63B illustrates replacing peristaltic pumps of FIG. 58B with usingvacuum chambers 806 on output sample bags to drive fluid through fluidiclines. FIG. 63A is identical to FIG. 62B in fluid line layout and inoperation of UFL 600 and MAG 123 with chambers 806, except that the UFLlarge entities output 6070 connects to large entities container 932 inblood bag form with small entities container 932 enclosed in vacuumchamber 806 replacing container 933 of FIG. 62B, and small entitiesoutput 6090 connects to connector 803.

FIG. 64A illustrates replacing peristaltic pumps of FIG. 59B with usingpressurized chambers 800 on input sample bags 928 and 929 to drive fluidthrough channel 201 of MAG 123. In FIG. 64A, pump 500, pump tubing504/505, and flow limiter 509/510 of FIG. 59B are removed. Channel 201is connected directly to T-connector 921. Sample liquid bag 928 and MAGbuffer bag 929 are each enclosed in a pressure chamber 800. Pressure inchambers 800 enclosing bags 928 and 929 are increased to force samplefluid or buffer fluid into channel 201 to start MAG 123 separation.After MAG 123 separation and sample fluid in bag 928 is depleted, bag934 and bag 9342 are each filled with either negative entities orpositive entities from MAG 123 after MAG separation.

FIG. 64B illustrates replacing peristaltic pumps of FIG. 59B with usingvacuum chambers 806 on output sample bags 934 and 9342 to drive fluidthrough channel 201 of MAG 123. FIG. 64B is same as FIG. 64A, exceptpressure chambers 800 are removed. Output sample bags 934 and 9342 areeach enclosed in a vacuum chamber 806. Vacuum in chambers 806 enclosingbags 934 and 9342 are increased to force entities sample from bag 928 orMAG buffer fluid from bag 929 to flow into channel 201 of MAG 123 tostart MAG 123 separation. After sample in bag 928 is depleted, and MAG123 separation finish, bags 934 and 9342 contain positive sample andnegative sample from MAG 123 channel 201 output.

FIG. 65A illustrates replacing peristaltic pumps of FIG. 60B with usingpressurized chambers 800 on sample liquid bag 928 and UFL buffer bag 931to drive fluid through UFL 600. In FIG. 65A, pump 500, pump tubing504/505, and flow limiter 509/510 of FIG. 60B are removed. Sample liquidbag 928 and UFL buffer bag 931 are each enclosed in a pressure chamber800. Pressure in chambers 800 enclosing bags 928 and 931 are increasedto force sample fluid and UFL buffer fluid into UFL 600 inlets to startUFL 600 separation. After UFL 600 separation, sample fluid in bag 928 isdepleted, bag 932 contains large entities fluid and bag 933 containssmall entities fluid.

FIG. 65B illustrates replacing peristaltic pumps of FIG. 60B with usingvacuum chambers 906 on output sample bags 932 and 933 to drive fluidthrough UFL 600. FIG. 65B is same as FIG. 65A, except pressure chambers800 are removed. Output sample bags 932 and 933 are each enclosed in avacuum chamber 806. Vacuum in chambers 806 enclosing bags 932 and 933are increased to force sample liquid from bag 928 and UFL buffer fluidfrom bag 931 to flow through UFL 600 to start UFL separation. Aftersample in bag 928 is depleted, and UFL 600 separation finish, bag 932contains large entities fluid and bag 933 contains small entities fluid.

Structures, components, and methods as described from FIG. 55A throughFIG. 65B on enclosed fluidic lines including one UFL 600 and one MAG123, may be applied to FIG. 47 through FIG. 52 without limitation, whereenclosed fluidic lines including multiple MAGs 123 and multiple UFLs 600may be achieved with replicating the components on single UFL 600 andsingle MAG 123 from FIG. 55A through FIG. 65B on each of the UFLs 600and MAGs 123 of FIG. 47 through FIG. 52.

FIG. 66 through FIG. 88 illustrate embodiments of process flows toutilize MAG and UFL devices to separate biological entities from variousbiological samples. For simplicity of description, terms UFL and MAG areused in these figures for explanation. However, UFL may be any of UFL600, 650, 620, 630, 640 of FIG. 40A, FIG. 41A, FIG. 42A, FIG. 43, whileMAG may be any of MAG 121, 122, 123, 124, 124,125, 126, 127, 128, 129with corresponding channel types as described in prior figures withoutlimitation and without sacrifice of performance. If a component, or astructure, in FIG. 66 through FIG. 88 shares same name as in priorfigures, it then means the same component, or same structure as in priorfigures.

FIG. 66 illustrates embodiment of a first process flow to separatebiological entities from peripheral blood using UFL and MAG. In step5801, peripheral blood sample is collected from a patient or personunder test; in step 5802, red blood cell lysing may be performed on saidperipheral blood sample, where step 5802 in another embodiment may beskipped; in step 5803, said blood sample from step 5802, or directlyfrom step 5801, is injected in UFL entity fluid inlet 602, while UFLbuffer fluid is injected in outlet 604; in step 504, set frequency andvibration strength of PZT attached to UFL to produce standing wave andpressure nodes in UFL fluid; in step 5805, UFL outlet 607 outputs targetsample that contains large size entities or cells; in step 5806, addinto target sample from step 5805 magnetic labels hybridized withantibodies or ligands, which specifically bind to surface antigens orreceptors on target cells or entities; in step 5807, target sample fromstep 5806 is incubated to form magnetic labels binding to target cellsor entities; in step 5808, flow target sample from step 5807 through MAGchannel at magnetic separation position, where during step 5808,negative MAG sample may be forwarded as in 5815 to be collected in step5813; in step 5809, target cells or entities bound with magnetic labelare separated by MAG within the MAG channel; in step 5810, after step5809, buffer fluid may be flown through MAG channel to wash out residuenon-target entities without magnetic label, the washed out fluid may beforwarded as in 5816 to be collected as negative MAG sample in step5813, where step 5810 may be skipped in another embodiment; in step5811, after step 5810 or directly after step 5809, separated entitiesconglomerate in MAG channel may be dissociated into isolated cells orentities; in step 5812, buffer fluid is flown through MAG channel towashed out dissociated cells and entities in MAG channel, which, asshown by 5817, may be collected as positive MAG sample in step 5814.

Peripheral blood sample of FIG. 66 may also be other body fluids,including but not limited to: saliva, tear, mucus, urine, secretion fromvarious organs of body.

FIG. 67 illustrates an embodiment of second process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 67 is same as FIG. 66, except step 5803, step 5804, and step5805 of FIG. 66 are removed between step 5802 and step 5806 in FIG. 67.While in FIG. 67, blood sample from step 5802, or blood sample directlyfrom step 5801, is centrifuged in step 6201 to extract target samplecontaining white blood cells. Target sample form step 6201 is then sentto step 5806, from step 5806 FIG. 67 flow is same as in FIG. 66.

FIG. 68 illustrates an embodiment of third process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 68 is same as FIG. 66, except step 5803, step 5804, and step5805 of FIG. 66 are removed between step 5802 and step 5806 in FIG. 68.While in FIG. 68, peripheral blood sample collected from patient orperson under test as in step 6301, which is same as step 5801 of FIG.66, is regarded target sample. Target sample form step 5802 after redblood cell lysing after step 6301, or directly from step 6301, is thensent to step 5806. From step 5806, FIG. 68 flow is same as in FIG. 66.

FIG. 69 illustrates an embodiment of fourth process flow to separatebiological entities from peripheral blood using MAG. Every other aspectof FIG. 69 is same as FIG. 66, except step 5801, step 5802, step 5803,step 5804, and step 5805 of FIG. 66 are removed before step 5806 in FIG.69. While in FIG. 69, target sample is collected after apheresis ofperipheral blood sample collected from patient or person under test.Target sample form step 6401 is then sent to step 5806. From step 5806,FIG. 69 flow is same as in FIG. 66.

FIG. 70 illustrates an embodiment of fifth process flow to separatebiological entities from tissue sample using UFL and MAG. Every otheraspect of FIG. 70 is same as FIG. 66, except step 5801, step 5802, andstep 5803 are removed before step 5804 in FIG. 70. In FIG. 70, tissuesample is collected in step 6501. In step 6502, tissue sample from step6501 is dissociated in a fluid base. In step 6503, dissociated tissuefluid of step 6502 is injected into UFL channel through inlet 602 andUFL buffer fluid is injected through inlet 604. From step 5804, FIG. 70flow is same as in FIG. 66. Tissue sample of FIG. 70 may include any of:human body tissue aspirate, human organ tissue aspirate, bone marrow,animal body or organ tissue aspirate. Target cells or entities of FIG.70 may be rare disease cells, for example cancer cells, ormicro-organisms, for example bacteria.

FIG. 71 illustrates an embodiment of sixth process flow to separatebiological entities from tissue sample using MAG. Every other aspect ofFIG. 71 is same as FIG. 70, except step 6503, step 5804, and step 5805are removed before step 5806 in FIG. 71. In FIG. 71, tissue sample fromstep 6501 is dissociated in a fluid base in step 6502 to form targetsample, and continues process in step 5806. From step 5806, FIG. 71 flowis same as in FIG. 70.

FIG. 72 illustrates an embodiment of seventh process flow to separatebiological entities from surface swab sample using UFL and MAG. Everyother aspect of FIG. 72 is same as FIG. 66, except step 5801, step 5802,and step 5803 are removed before step 5804 in FIG. 72. In FIG. 72,surface entities are collected in step 6701 by swab. In step 6702,surface entities collected on swab are dissolved in a fluid base. Instep 6703, fluid base with dissolved surface entities from step 6702 isinjected into UFL channel through inlet 602 and UFL buffer fluid isinjected through inlet 604. From step 5804, FIG. 72 flow is same as inFIG. 66. Surface entities of FIG. 72 may be collected by swab fromsubjects including any of: human body, saliva, body fluid, human bodydischarge, animal, plant, soil, air, water, and merchandise. Targetcells or entities of FIG. 72 may include cells from human body, oranimal body, or plant, or include micro-organisms, for example bacteria,mold, or spores.

FIG. 73 illustrates an embodiment of eighth process flow to separatebiological entities from surface swab sample using MAG. Every otheraspect of FIG. 73 is same as FIG. 72, except step 6703, step 5804, andstep 5805 are removed before step 5806 in FIG. 73. In FIG. 73, surfaceentities collected on swab in step 6701 are dissolved in a fluid base instep 6702 to form target sample, and continues process in step 5806.From step 5806, FIG. 73 flow is same as in FIG. 72.

FIG. 74 illustrates an embodiment of ninth process flow to separatebiological entities from solid sample using UFL and MAG. Every otheraspect of FIG. 74 is same as FIG. 66, except step 5801, step 5802, andstep 5803 are removed before step 5804 in FIG. 74. In FIG. 74, solidsample is collected in step 6901. In step 6902, solid sample from step6901 is dissociated in a fluid base. In step 6903, dissociated solidsample fluid of step 6902 is injected into UFL channel through inlet 602and UFL buffer fluid is injected through inlet 604. From step 5804, FIG.74 flow is same as in FIG. 66. Tissue sample of FIG. 70 may include anyof: solid biological products or waste generated by human, animal, orplant, powder, and soil. Target cells or entities of FIG. 74 may includecells from human body, or animal body, or plant, or includemicro-organisms, for example bacteria, mold, or spores.

FIG. 75 illustrates an embodiment of tenth process flow to separatebiological entities from solid sample using MAG. Every other aspect ofFIG. 75 is same as FIG. 74, except step 6903, step 5804, and step 5805are removed before step 5806 in FIG. 75. In FIG. 75, solid sample fromstep 6901 is dissociated in a fluid base in step 6902 to form targetsample, and target sample is continuously processed in step 5806. Fromstep 5806, FIG. 75 flow is same as in FIG. 74.

FIG. 76A illustrates addition of both magnetic and fluorescent labelsinto fluid samples for specific binding to target cells or entities.FIG. 76A shows that step 5806 of FIG. 66 through FIG. 75 may be modifiedto step 58061, where in addition to magnetic labels, fluorescent labelshybridized with antibodies or ligands, which specifically bind tosurface antigens or receptors on target cells or entities, may also beadded in target sample from step 5805.

FIG. 76B then illustrates that incubation step 5807 of FIG. 66 throughFIG. 75 may also be modified to step 58071, which includes incubation ofboth magnetic and fluorescent labels at the same time to form specificbinding to target cells or entities. Binding sites of magnetic labelsand fluorescent labels on same target cells or entities may bedifferent.

Steps 5806 and step 58061 may be realized in a flow connector includingany one of 801, 802, 803, 8010, 8020, 8030 of prior figures, where flowconnector may contain pre-filled hybridized magnetic labels andfluorescent labels in liquid solution, or in dry powder form. Step 5807and step 58071 may also occur in said flow connector, where said flowconnector may also be located in a temperature control chamber tocontrol incubation speed and quality. In another embodiment, said flowconnector may have attached or embedded temperature control circuit tocontrol incubation in flow connector.

FIG. 77A illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL before magnetic separation by MAG. FIG. 77Ashows that for each of FIG. 66 through FIG. 75, step 5818 and step 5819may be added between step 5807 and step 5808. After target sample isincubated in step 5807, in step 5818, target sample may be injected intosecond UFL through inlet 602, and buffer fluid may be injected intosecond UFL through inlet 604. In step 5819, second UFL outputs targetsample containing large entities from outlet 607, and non-bound freemagnetic labels are output from second UFL outlet 609. Then in step5808, target sample containing large entities from second UFL outlet 607is passed through MAG channel for magnetic separation. Target sample instep 5819 may contain cells 10/30 or entities bound with magneticlabels. Second UFL having an attached PZT that operates with a specifiedultrasound vibration amplitude and frequency to create standing wave insecond UFL channel fluid is assumed in step 5819.

FIG. 77B illustrates process of removing non-bound free magnetic labelsfrom sample fluid by UFL after magnetic separation by MAG. FIG. 77Bshows that for each of FIG. 66 through FIG. 75, step 5820 and step 5821may be added between step 5812 and step 5814, replacing path 5817. Aftermagnetic conglomerate within MAG channel is dissociated and the positiveMAG sample entities from MAG channel are flushed out as in step 5812,flushed out positive MAG sample may be injected into third UFL throughinlet 602, and buffer fluid may be injected into third UFL through inlet604. In step 5821, third UFL outputs positive MAG sample containinglarge entities from outlet 607, and non-bound free magnetic labels areoutput from third UFL outlet 609. Then in step 5814, positive MAG samplewith reduced or depleted free magnetic labels may be collected. ThirdUFL having an attached PZT that operates with a specified ultrasoundvibration amplitude and frequency to create standing wave in third UFLchannel fluid is assumed in step 5821.

FIG. 78A illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL before magneticseparation by MAG. FIG. 78A is similar as FIG. 77A, with replacing step5807 of FIG. 77A with step 58071 of FIG. 76B, and replacing step 5819with step 58191. After adding magnetic labels and fluorescent labelsinto target sample as in step 58061 of FIG. 76A, target sample isincubated in step 58071 same as in FIG. 76B to form magnetic label andfluorescent label binding to target cells or entities. In step 5818,target sample may be injected into second UFL through inlet 602, andbuffer fluid may be injected into second UFL through inlet 604. In step58191, second UFL outputs target sample containing large entities fromoutlet 607, and non-bound free magnetic labels and free fluorescentlabels are output from second UFL outlet 609. Then in step 5808, targetsample containing large entities from second UFL outlet 607 is flownthrough MAG channel for magnetic separation. Target sample in step 58191may contain cells 30 or entities bound with magnetic and fluorescentlabels. Second UFL having an attached PZT that operates with a specifiedultrasound vibration amplitude and frequency to create standing wave insecond UFL channel fluid is assumed in step 58191.

FIG. 78B illustrates process of removing non-bound free magnetic labelsand free fluorescent labels from sample fluid by UFL after magneticseparation by MAG. FIG. 78B is similar as FIG. 77B, with replacing step5821 of FIG. 77A with step 58211. Separated entities in step 5812 andstep 5820 of FIG. 78B may contain: cells 30 or entities bound withmagnetic and fluorescent labels, non-bound free magnetic labels, andsmall amount of non-bound free fluorescent labels due to non-specificbinding to conglomerate in MAG channel during magnetic separation. Instep 58212, third UFL outputs positive MAG sample containing largeentities from outlet 607, and non-bound free magnetic and free opticallabels are output from third UFL outlet 609. Then in step 5814, positiveMAG sample with reduced or depleted free magnetic labels and freefluorescent labels may be collected. Third UFL having an attached PZTthat operates with a specified ultrasound vibration amplitude andfrequency to create standing wave in third UFL channel fluid is assumedin step 58212.

FIG. 79 illustrates continued process of negative MAG sample after MAGseparation, as in step 408 of FIG. 31, through UFL to remove smallentities and passing of large entities into various cell processingdevices and procedures. Step 5813 is same as in FIG. 66 through FIG. 75,where negative MAG sample is collected during MAG separation of a targetsample. In step 5822, negative MAG sample of step 5813 is injected intofourth UFL inlet 602, and UFL buffer is injected into inlet 604 offourth UFL. In step 5823, fourth UFL outputs negative MAG samplecontaining large entities from outlet 607, and small size entities areremoved from large entities and output from fourth UFL outlet 609, a PZTthat attaches to fourth UFL and operates with a specified ultrasoundvibration amplitude and frequency to create standing wave in fourth UFLis assumed. Finally, negative MAG sample containing large entities fromoutlet 607 of fourth UFL may be sent to be analyzed by any of: cellcounter 903, cell imager 904, flow cytometer or sorter 905, DNA/RNAsequencer 906. Alternatively, output from cell counter 903, or outputfrom cell imager 904, or output from flow cytometer or sorter 905, maybe further sent to be processed by DNA/RNA sequencer 906 as indicatedrespectively by path 936, 946, and 956. Negative MAG sample containinglarge entities from outlet 607 of fourth UFL in step 5823 may also besent into the process of cell genetic modification and cell expansion5824. Prior to DNA/RNA sequencing in DNA/RNA sequencer 906, a polymerasechain reaction (PCR) procedure on DNA/RNA sample obtained from celllysing of large size entities from outlet 607 of fourth UFL from step5823 may be performed, where PCR may be targeting one or more targetDNA/RNA sequences and amplifies the number of target DNA/RNA sequencesin the DNA/RNA sample.

FIG. 80 illustrates continued process of negative MAG sample after MAGseparation, as in step 408 of FIG. 31, through UFL to retrieve smallentities and passing of small entities into various molecule or smallentity processing devices. After step 5813 of FIG. 66 through FIG. 75,where negative MAG sample is collected during MAG separation of a targetsample, in step 5822, negative MAG sample of step 5813 is injected intofourth UFL inlet 602, and UFL buffer is injected into inlet 604 offourth UFL. In step 5825, fourth UFL outputs negative MAG samplecontaining large entities from outlet 607, and small size entitiesincluding DNA, RNA, molecules, and other small particles are output fromfourth UFL outlet 609, a PZT that attaches to fourth UFL and operateswith a specified ultrasound vibration amplitude and frequency to createstanding wave in fourth UFL is assumed. Finally, small size entitiesfrom outlet 609 of fourth UFL may be sent to be analyzed by any of:particle counter 5835, particle imager 5836, flow cytometer or sorter905, DNA/RNA sequencer 906. Alternatively, output from particle counter5835, or output from particle imager 5836, or output from flow cytometeror sorter 905, may be further sent to be processed by DNA/RNA sequencer906 as indicated respectively by path 5827, 5828, and 956. DNA/RNAsequencer 906 may contain a PCR step on small size entities from outlet609 of fourth UFL from step 5825 prior to DNA/RNA sequencing, where PCRmay target one or more particular DNA/RNA sequences to amplify inquantity.

FIG. 81 illustrates entities analysis of negative MAG sample after MAGseparation, as in step 407 of FIG. 31, into various analyzing devices.After step 5813 of FIG. 66 through FIG. 75, where negative MAG sample iscollected during MAG separation of a target sample, collected negativeMAG sample may be sent to be analyzed by any of: cell counter 903, cellimager 904, flow cytometer or sorter 905, particle counter 5835,particle imager 5836, DNA/RNA sequencer 906. Alternatively, output fromcell counter 903, or output from cell imager 904, or output from flowcytometer or sorter 905, or output from particle counter 5835, or outputfrom particle imager 5836, may be further sent to be processed byDNA/RNA sequencer 906 as indicated respectively by path 936, 946, 956,5827, and 5828. Negative MAG sample may also be sent into the process ofcell genetic modification and cell expansion 5824. DNA/RNA sequencer 906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing ofcells contained within negative MAG sample; and (2) DNA/RNA/moleculescontained within negative MAG sample. Prior to DNA/RNA sequencing, PCRmay target one or more particular DNA/RNA sequences to amplify inquantity.

FIG. 82 illustrates continued process of positive MAG sample after MAGseparation, as in step 408 of FIG. 31, through UFL to remove smallentities and passing of large entities into various cell processingdevices and procedures. Step 5814 is same as in FIG. 66 through FIG. 75,where positive MAG sample is collected after MAG separation of a targetsample. In step 5829, positive MAG sample of step 5814 is injected intofifth UFL inlet 602, and UFL buffer is injected into inlet 604 of fifthUFL. In step 5830, fifth UFL outputs positive MAG sample containinglarge entities from outlet 607, and small size entities are removed fromlarge entities and output from fifth UFL outlet 609, a PZT that attachesto fourth UFL and operates with a specified ultrasound vibrationamplitude and frequency to create standing wave in fifth UFL is assumed.Finally, positive MAG sample containing large entities from outlet 607of fifth UFL may be sent to be analyzed by any of: cell counter 903,cell imager 904, flow cytometer or sorter 905, DNA/RNA sequencer 906.Alternatively, output from cell counter 903, or output from cell imager904, or output from flow cytometer or sorter 905, may be further sent tobe processed by DNA/RNA sequencer 906 as indicated respectively by path936, 946, and 956. Positive MAG sample containing large entities fromoutlet 607 of fifth UFL in step 5830 may also be sent into the processof cell genetic modification and cell expansion 5824. DNA/RNA sequencer906 may contain a PCR step on DNA/RNA obtained after cell lysing oflarge size entities from outlet 607 of fifth UFL from step 5830 prior toDNA/RNA sequencing, where PCR may target one or more particular DNA/RNAsequences to amplify in quantity.

FIG. 83 illustrates continued process of positive MAG sample after MAGseparation, as in step 408 of FIG. 31, through UFL to retrieve smallentities and passing of small entities into various molecule or smallentity processing devices. After step 5814 of FIG. 66 through FIG. 75,where positive MAG sample is collected after MAG separation of a targetsample, in step 5829, positive MAG sample of step 5814 is injected intofifth UFL inlet 602, and UFL buffer is injected into inlet 604 of fifthUFL. In step 5831, fifth UFL outputs positive MAG sample containinglarge entities from outlet 607, and small size entities including DNA,RNA, molecules, and other small particles bound by magnetic labels areoutput from fifth UFL outlet 609, a PZT that attaches to fifth UFL andoperates with a specified ultrasound vibration amplitude and frequencyto create standing wave in fifth UFL is assumed. Finally, small sizeentities from outlet 609 of fifth UFL may be sent to any of: particlecounter 5835, particle imager 5836, flow cytometer or sorter 905,DNA/RNA sequencer 906. Alternatively, output from particle counter 5835,or output from particle imager 5836, or output from flow cytometer orsorter 905, may be further sent to be processed by DNA/RNA sequencer 906as indicated respectively by path 5827, 5828, and 956. DNA/RNA sequencer906 may contain a PCR step on small size entities from outlet 609 offifth UFL from step 5831 prior to DNA/RNA sequencing, where PCR maytarget one or more particular DNA/RNA sequences to amplify in quantity.

FIG. 84 illustrates entities analysis of positive MAG sample after MAGseparation, as in step 407 of FIG. 31, into various analyzing devices.After step 5814 of FIG. 66 through FIG. 75, where positive MAG sample iscollected after MAG separation of a target sample, collected positiveMAG sample may be sent to be analyzed by any of: cell counter 903, cellimager 904, flow cytometer or sorter 905, particle counter 5835,particle imager 5836, DNA/RNA sequencer 906. Alternatively, output fromcell counter 903, or output from cell imager 904, or output from flowcytometer or sorter 905, or output from particle counter 5835, or outputfrom particle imager 5836, may be further sent to be processed byDNA/RNA sequencer 906 as indicated respectively by path 936, 946, 956,5827, and 5828. Positive MAG sample may also be sent into the process ofcell genetic modification and cell expansion 5824. DNA/RNA sequencer 906may contain a PCR step on: (1) DNA/RNA obtained after cell lysing ofcells contained within positive MAG sample; and (2) DNA/RNA/moleculescontained within positive MAG sample, prior to DNA/RNA sequencing, wherePCR may target one or more particular DNA/RNA sequences to amplify inquantity.

FIG. 85A illustrates adding fluorescent labels to specifically bind totarget entities within negative MAG sample immediately after negativeMAG sample collection. FIG. 85A shows that in step 58131, immediatelyafter step 5813, where negative MAG sample is collected during MAGseparation, fluorescent labels which are hybridized with antibodies orligands, and specifically bind to surface antigens or receptors ontarget cells or entities are added into the negative MAG sample, andthen negative MAG sample is incubated to form fluorescent labels bindingto target cells or entities. Step 58131 may be inserted in FIG. 79 andFIG. 80 between step 5813 and step 5822, or inserted in FIG. 81immediately after step 5813 and before devices or processes 903, 904,905, 906, 5824, 5825, and 5826.

FIG. 85B illustrates adding fluorescent labels to specifically bind totarget entities within positive MAG sample immediately after positiveMAG sample collection. FIG. 85B shows that in step 58141, immediatelyafter step 5814, where positive MAG sample is collected after MAGseparation, fluorescent labels which are hybridized with antibodies orligands, and specifically bind to surface antigens or receptors ontarget cells or entities are added into the positive MAG sample, andthen positive MAG sample is incubated to form fluorescent labels bindingto target cells or entities. Step 58141 may be inserted in FIG. 82 andFIG. 82 between step 5814 and step 5829, or inserted in FIG. 84immediately after step 5814 and before devices or processes 903, 904,905, 906, 5824, 5825, and 5826.

FIG. 86A through FIG. 93 describe methods to achieve pre-symptom earlystage tumor detection, especially in asymptomatic tumor patients who arein very early stage, or have not been diagnosed with tumor, or showingno symptom, or tumor is in such infancy or early stage that may not bedetected or located by conventional methods, including imaging or bloodtest. In the description, terms of “cancer” and “tumor” may be usedinterchangeably for same meaning.

Malignant tumor, or cancer, is a disease that results from geneticmutation of normal body cells, which become astray from original cellfunctions, multiplying fast, and evading normal cell life cycle ofprogrammed cell death by human immune system. To increase the survivalchance of a patient carrying cancer, it is imperative to identify andlocate the cancer at as earliest stage as possible. In state-of-artmedicine practiced today, tumors are still found or identified eitherafter physical identification by imaging methods including ultrasound,X-ray, computerized tomography (CT), magnetic resonance imaging (MM),and in most cases after patient showing symptoms due to cancer growth.For cancer to be identified by imaging methods or by patient showingsymptoms, cancer growth is typically already underway, and in most caseswell developed with cancer cells in the body already growing insignificant numbers. For certain cancers, for example pancreatic cancerwhich is typically asymptomatic even in late stages, detection byconventional method would usually be too late to provide meaningfulmedical intervention. It is desirable, and imperative, to have a cancerdetection method that is able to detect occurrence of a cancer, withlocation of origination, at its infancy stage, where such detection ispreferred to before cancer's significant growth, and before anystatistical possibility of cancer's spreading from a local growth toother parts of body. This detection method is desirable to beadministered to a person-under-test through conventional clinical means,for example typical peripheral blood collection and blood test. Byachieving pre-symptom early stage cancer detection and knowing withconfidence of cancer type and location, medical intervention may providemost effectiveness in removal of cancer cells, significantly increasesurvival rate, and eventually cure of cancer, and at the same timesignificantly reducing financial and social burden of cancer treatment.

FIG. 86A illustrates first example of cancer treatment. Cancer cell 2002exhibits surface antigen, ligand or surface marker 2004, which can beused to identity and kill cancer cell 2002 in human body by an immunecell 2001 with a surface anti-body, or receptor 2003 that specificallybinds to surface marker 2004, as shown by 2034. Immune cell 2001 may beextracted from the person-under-test (PUT), or from a donor person.Immune cell 2001 may be genetically modified after being collected fromthe PUT or donor to express surface receptor 2003. Immune cell 2001 withantibody 2003 may be expanded or cultivated in ex vivo environment.Immune cell 2001 may be engineered to suppress, or evade, immune systemresponse of the PUT if immune cell is collected from donor. In practice,immune cells 2001 with antibody 2003 may be administered to PUT withblood infusion, where immune cells 2001 will then find and bind tocancer cells 2002 in vivo through antibody-antigen bond 2034 betweenantibody 2003 and marker 004 and kill the cancer cell 2002. For example,cell 2001 may be a type of ex vivo engineered chimeric antigen receptorT cell (CAR-T) with 2003 being chimeric antigen receptor (CAR) thattargets cancer cell 2001 which has a surface marker 2004. Receptor 2003may be any of, but not limited to, CD19, CD20, CD22, CD30, ROR1, κ lightchain, CD123, CD33, CD133, CD138, and B-cell maturation antigen.

FIG. 86B illustrates second example of cancer treatment. In PUT, theperson's internal immune cell 2007, for example a T cell, functions toidentify cells which need to be terminated as a normal cell life cycle.For a normal cell, immune cell 2007 forms receptor 2051 to antigen 2052bond, which enables immune cell 2007 to terminate a normal cell at endof life cycle of the normal cell. Receptor 2501 may include T-cellreceptor (TCR), CD 28. However, cancer cell 2002 evades such programmeddeath from immune cell 2007 by forming another ligand 2054 and receptor2053 bond with immune cell 2007, which effectively disables the receptor2051 to antigen 2052 bond that functions to terminate the cancer cell2002, for example ligand 2054 and receptor may be PD-L1 and PD-1respectively. In FIG. 86B method, antibody 2056 or antigen 2055 may beadministered to PUT, such that antibody 2056 may bind to ligand 2054 andantigen 2055 may bind to receptor 2053 in vivo of PUT body, causingeffective disconnection of 2053 to 2054 bond, and making termination ofthe cancer cell 2002 by the immune cell 2007 through 2051-2052 bondbeing possible.

FIG. 86C illustrates third example of cancer treatment. In FIG. 86Cmethod, part of immune system cells, for example dendritic cell 2008,may be inserted with cancer cell 2002 RNA or antigen ex vivo, so thatcell 2008 may express cancer 2008 surface antigen 2004. When dendriticcell 2008 with surface antigen 2004 is injected into PUT as in 2009,immune cell 2007 of PUT, for example T cell, may be trained or directedas in 2010 by the dendritic cell 2008 to recognize the expressed cancerantigen 2004 with expressing corresponding receptor 2003 on immune cell2007. Immune cell 2007 then is able to recognize cancer cell 2002 in PUTwith receptor 2003 to antigen 2004 binding 2034, and subsequentlyterminate cancer cell 2002.

During termination of cancer cell 2002 in FIG. 86A, FIG. 86B and FIG.86C, lysis of cancer cell 2002 occurs and genetic material, includingtumor DNA and RNA, within cancer cell 2002 is released and will finallyenter blood stream of PUT. In this invention, cell 2001 with receptor2003 of FIG. 86A, antibody 2056 and antigen 2055 of FIG. 86B, cell 2008with antigen 2004 of FIG. 86C, will be categorially referred to as“anti-tumor agent”, which may be administer externally to a PUT andcausing a target type of cancer cell 2002, if existing in PUT, toterminate and release genetic material into blood stream of PUT.

FIG. 87 illustrates first method of this invention to achievepre-symptom early stage tumor detection, which includes the sequentialsteps of: (step 1001) administer anti-tumor agent to a person-under-test(PUT) to cause termination and lysis of target tumor cells, if existingin PUT, and release genetic material into blood stream of PUT; (step1002) collect peripheral blood from PUT; (step 1003) obtain cell-freeplasma from the collected peripheral blood; (step 1004) perform PCR onthe cell-free plasma to amplify quantity of known DNA or RNA sequencesthat identify target tumor cells, resulting in PCR sample; (step 1005)perform DNA or RNA sequencing on PCR sample and ascertain existence ofknown DNA or RNA sequence that identifies target tumor cells.

Step 1001 anti-tumor agent may be one of, or a combination of,anti-tumor agents as described in FIG. 86A, FIG. 86B and FIG. 86C.Anti-tumor agent may be in sufficiently small amount that does noteliminate target tumor cells in PUT, but will cause lysis of a pluralityof target tumor cells to release genetic material to be collected instep 1003, amplified in step 1004 and detected in step 1005. Withanti-tumor agent being in sufficiently small amount, adverse effects ofFIG. 86A, FIG. 86B and FIG. 86C process, including cytokine releasesyndrome, neurotoxicity, or off-tumor aplasia, may be limited to notcause clinical conditions of PUT requiring medical attention. FIG. 87method implies that the DNA or RNA sequence of step 1004 and step 1005is generic to the type of target tumor cells and is independent of PUT.

PUT of FIG. 87 may be a person without prior history of tumor, orwithout showing symptom of tumor, or without showing any physical signor results of tumor through conventional medical examination methods.PUT may be a person at risk of target tumor, for example due to geneticmutation, family history, age, environment, or occupation. PUT may be aperson who has been treated for target tumor, but needing monitor ofrecurrence of target tumor. PUT may or may not carry target tumor cells.In the case that step 1005 confirms existence of known DNA or RNAsequence that identifies target tumor cells, it may be concluded thatPUT carries target tumor, where the amount of DNA or RNA detected instep 1005 may be used to project stage and severity of tumor in PUT. Dueto the specificity of known DNA or RNA sequence that identifies targettumor, existence of such DNA or RNA may also confirm simultaneously mostprobable location of occurrence of such tumor when PUT is a pre-symptomand very early stage patient. In the case that step 1005 does not detectknown DNA or RNA sequence that identifies target tumor cells, or doesnot detect such DNA or RNA sequence at an amount that is above aconfidence threshold value, it may be concluded that PUT does not carrytarget tumor.

In practice, a time lapse may be needed after administering anti-tumoragent in step 1001, and before tumor genetic material may be releasedinto blood stream of PUT after tumor cell lysis for collection in step1002. Therefore, a scheduled waiting period, or a peripheral bloodcollection time window, may be implemented between step 1001 and step1002. Said scheduled waiting period may be between 15 minutes to 30minutes in one embodiment, between 30 minutes to 1 hour in anotherembodiment, between 30 minutes to 1 hour in yet another embodiment,between 1 hour to 2 hours in yet another embodiment, between 2 hours to6 hours in yet another embodiment, between 6 hours to 12 hours in yetanother embodiment, between 12 hours to 24 hours in yet anotherembodiment, between 1 day to 2 days in yet another embodiment, between 2days to 4 days in yet another embodiment, between 4 days to 10 days inyet another embodiment, between 10 days to 15 days in yet anotherembodiment, and between 15 days to 30 days in yet another embodiment.Said collection time window has a start time and an end time after thetime of said administering of said anti-tumor agent, where the starttime and end time may be between 15 minutes to 30 minutes in oneembodiment, between 30 minutes to 1 hour in another embodiment, between30 minutes to 1 hour in yet another embodiment, between 1 hour to 2hours in yet another embodiment, between 2 hours to 6 hours in yetanother embodiment, between 6 hours to 12 hours in yet anotherembodiment, between 12 hours to 24 hours in yet another embodiment,between 1 day to 2 days in yet another embodiment, between 2 days to 4days in yet another embodiment, between 4 days to 10 days in yet anotherembodiment, between 10 days to 15 days in yet another embodiment, andbetween 15 days to 30 days in yet another embodiment. Additionally, amultiple-cycled repeated step 1002 to step 1005 process may be performedas shown by procedure 1009, where procedure 1009 may include a scheduledwaiting period, such that existence of target tumor cells in PUT may bemonitored and ascertained during an extended amount of time for a morecomplete anti-tumor agent action on target tumor cells. Said scheduledwaiting period in procedure may be between 6 hours to 12 hours in oneembodiment, between 12 hours to 24 hours in another embodiment, between1 day to 2 days in yet another embodiment, between 2 days to 4 days inyet another embodiment, between 4 days to 10 days in yet anotherembodiment, between 10 days to 15 days in yet another embodiment, andbetween 15 days to 30 days in yet another embodiment.

Advantages of FIG. 87 method are: (1) PUT may be a person carrying tumorat early stage but without tumor indication in conventional tests, thusenabling pre-symptom early stage cancer intervention; (2) byadministering anti-tumor agent targeting a specific tumor, for examplebreast cancer, pancreatic cancer, lung cancer, detection ofcorresponding tumor DNA or RNA signal also confirms type and origin ofsuch cancer enabling fast and targeted treatment; (3) by administeringanti-tumor agent targeting a specific tumor, released DNA or RNA inblood stream may spike in a well-defined time window afterwards, whichprovides an enhanced signal-to-noise ratio (SNR) of tumor DNA or RNAdetection and may enable high sensitivity and high accuracy detectioneven when the actual amount of tumor cells in PUT is still at much fewerthan being detectable by conventional tests; (4) this method may allow apanel of multiple anti-tumor agents targeting multiple types of tumorsbeing applied simultaneously to PUT to detect existence of multipletypes of target tumors at the same time, as described in FIG. 89.Although FIG. 87 method targets pre-symptom early stage tumor, it ispossible to implement same method against dormant tumor, which may notshow fast growth, but has genetic mutation that has high risk ofmalignancy.

FIG. 88 illustrates a second method of tumor detection. FIG. 88 processis same as FIG. 87 in every other aspects, except that: after step 1001,instead of collecting peripheral blood as in step 1002 of FIG. 87, step1006 of FIG. 88 collects body fluid from around organ of PUT wheretarget tumor may occur, such body fluid would be where released DNA orRNA by lysed target tumor cells may be found, for example urine forbladder cancer, or prostate secretion for prostate cancer; then in step1007 replacing step 1003 of FIG. 87, obtain cell-free fluid from thecollected body fluid of step 1006; and then in step 108 replacing step1004 of FIG. 87, perform PCR on cell-free fluid to amplify quantity ofknown DNA or RNA sequences that identify target tumor cells, resultingin PCR sample. PCR sample of step 1008 then undergoes same step 1005 asin FIG. 87.

FIG. 89 illustrates a third method of tumor detection. FIG. 89 processis same as FIG. 87, but expanding tumor detection from one target tumorto multiple types of tumor. FIG. 89 process includes the sequentialsteps of: (step 3001) administer anti-tumor agents to PUT to cause lysisof a plurality types of target tumor cells to release genetic materialinto blood stream of PUT; (step 1002) collect peripheral blood from PUT;(step 1003) obtain cell-free plasma from the collected peripheral blood;(step 3004) perform PCR on the cell-free plasma to amplify quantity ofknown DNA or RNA sequences that identify each type of the pluralitytypes of target tumor cells, resulting in PCR sample; (step 3005)perform DNA or RNA sequencing on PCR sample and ascertain existence ofknown DNA or RNA sequence that identifies each type of the pluralitytypes of target tumor cells. In FIG. 89, ascertaining of existence ofany of the multiple types of tumors may be performed at same time inPUT. Anti-tumor agents in step 3001 may be one of anti-tumor agents asdescribed in FIG. 86A, FIG. 86B and FIG. 86C, which lyses multiple typesof tumors simultaneously. Anti-tumor agents in step 3001 may be acombination of more than one anti-tumor agents as described in FIG. 86A,FIG. 86B and FIG. 86C, where different anti-tumor agent lyses one type,or a sub-set of types, of the plurality types of tumors. Steps 1002 and1003 of FIG. 89 may be replaced by steps 1006 and 1007 of FIG. 88 tocollect body fluids, where step 3004 may correspondingly be updated withperforming PCR on cell-free fluid from step 1007.

FIG. 90, FIG. 91, and FIG. 92 illustrate embodiments of process flows toutilize MAG and UFL devices to obtain cell-free plasma from peripheralblood. For simplicity of description, terms UFL and MAG are used inthese figures for explanation. However, UFL may be any of UFL 600, 620,630, 640 of FIG. 40A, FIG. 41A, FIG. 42A, FIG. 43, while MAG may be anyof MAG 121, 122, 123, 124, 124,125, 126, 127, 128, 129 withcorresponding channel types as described in prior figures withoutlimitation and without sacrifice of performance.

FIG. 90 illustrates embodiment of first process flow to obtain cell-freeplasma of step 1003 from collected peripheral blood as in step 1002.After collection of peripheral blood in step 1002, peripheral blood maybe centrifuged as in step 5001, and result of centrifuge may containcell-free plasma that may directly achieve step 1003 as indicated bypath 5005. Alternatively, after centrifuge step 5001, blood plasma maybe depleted of certain blood cells, for example red blood cells, but maynot have enough purity for later stage PCR process. Plasma from step5001 may then be sent as in 5008 to undergo a UFL separation in step5002, where UFL large entities output contains any remaining cells inplasma, while UFL small entities output contains DNA or RNA and may bemore concentrated than plasma from step 5001. In yet another alternativepath, peripheral blood from step 1002 may skip step 5001 but directlyinput into UFL separation step 5002 as shown by path 5007, where the UFLseparates cells through large entities output while maintaining DNA andRNA in small entities output. Small entities output from UFL separationof step 5002 may then be used towards step 1003 as cell-free plasma asin path 5009. Further alternatively, magnetic labels hybridized withantibodies or ligands may be added to the plasma from UFL small entitiesoutput of step 5002 to bind to DNA or RNA within the plasma as in step5003. MAG device may be used to separate the DNA and RNA bound with themagnetic labels in step 5004, and the resulting positive MAG samplecontaining DNA and RNA then may be regarded the cell-free plasma of step1003.

FIG. 91 illustrates embodiment of second process flow to obtaincell-free plasma of step 1003 from collected peripheral blood as in step1002. After collection of peripheral blood in step 1002, peripheralblood may be centrifuged as in step 5001, and resulting blood plasma maybe depleted of certain blood cells, for example red blood cells. Then instep 5003, magnetic labels hybridized with antibodies or ligands may beadded to the plasma from step 5001 to bind to DNA or RNA within theplasma. Alternatively, peripheral blood from step 1002 may be useddirectly in step 5003 without step 5001 centrifuge as shown by path5015, and magnetic labels bind to the DNA or RNA in peripheral blood instep 5003. After step 5003, MAG device may be used to separate the DNAand RNA bound with the magnetic labels in step 5004, and the resultingpositive MAG sample containing DNA and RNA may be regarded the cell-freeplasma of step 1003 as indicated by path 5013. Further alternatively,positive MAG sample of step 5004 may undergo a UFL separation as in step5002, where UFL large entities output contains any remaining cells inplasma, while UFL small entities output contains DNA or RNA that arebound with magnetic labels. Small entities output from UFL separation ofstep 5002 may then be used towards step 1003 as cell-free plasma as inpath 5017.

FIG. 92 illustrates embodiment of third process flow to obtain cell-freeplasma of step 1003 from collected peripheral blood as in step 1002.After collection of peripheral blood in step 1002, peripheral blood maybe centrifuged as in step 5001, and resulting blood plasma may bedepleted of certain blood cells, for example red blood cells. Then instep 5023, magnetic labels hybridized with antibodies or ligands may beadded to the plasma from step 5001 to bind to cells within the plasma.Alternatively, peripheral blood from step 1002 may be used directly instep 5003 without step 5001 centrifuge as shown by path 5015, andmagnetic labels bind to the cells in peripheral blood in step 5023.After step 5023, MAG device may be used to separate the cells bound withthe magnetic labels in step 5024 from the plasma that contains DNA orRNA, and the resulting negative MAG sample containing DNA and RNA may beregarded the cell-free plasma of step 1003 as indicated by path 5013.Alternatively, negative MAG sample of step 5024 may undergo a UFLseparation as in step 5002, where UFL large entities output contains anyremaining cells in plasma from step 5012, while UFL small entitiesoutput contains DNA or RNA. Small entities output from UFL separation ofstep 5002 may then be used towards step 1003 as cell-free plasma as inpath 5017.

FIG. 93 illustrates a method to extend method of FIG. 89 for anti-agingpurpose. For conditions relating to aging, for example Alzheimer'sDisease, dementia, osteoporosis, and arthritis, human growth hormone orother anti-aging agent may be used to help cell growth to delay oralleviate symptoms occurrence of the conditions. For aging in general,human growth hormone or other anti-aging agents may help improve overallbody conditions due to tissue or cell replenishment. However, in use ofsuch anti-aging agent, a possible limitation is increased risk of tumoroccurrence. Due to the nature of tumor cells being fast growth andevading cell death by immune system, administering human growth hormoneor anti-aging agent in presence of any tumor, especially pre-symptomtumor or dormant tumor, such hormone or agent may incur growth of thesetumor cells. Thus, for low risk implementation of human growth hormoneor anti-aging agent, a tumor free condition of PUT is desired. FIG. 93initial flow steps of 3001, 1002, 1003, 3004 and 3005 are identical toFIG. 89, where existence of any of the multiple types of target tumorsin PUT may be ascertained in step 3005. In FIG. 93, after step 3005, inthe case that at least one type of tumor is confirmed to exist in PUT asin judgement 4001, corresponding tumor treatment may be performed instep 4002. After step 4002, another cycle of process from step 3001 tostep 3005 may be performed to ascertain tumor absence after treatment ofstep 4002. In the case that step 3005 finds no tumor existence as injudgement 4003, growth hormone or anti-aging agent may be administeredto the PUT as in step 4004. After step 4004, another cycle of step 3001through step 3005 may be performed to ascertain no tumor was promoted bythe step 4004. Flow from step 3001 to step 4004 may be repeated as manycycles as needed to achieve aging related conditions treatment goal.

While the current invention has been shown and described with referenceto certain embodiments, it is to be understood that those skilled in theart will no doubt devise certain alterations and modifications theretowhich nevertheless include the true spirit and scope of the currentinvention. Thus the scope of the invention should be determined by theappended claims and their legal equivalents, rather than by examplesgiven.

What is claimed is:
 1. A device for separating biological entities withmagnetic labels from a fluid sample, the device comprising: a first softmagnetic pole having a first base end contacting a first magnet, and afirst tip end smaller than said first base end, thereby concentratingmagnetic flux generated by said first magnet to said first tip end; asecond soft magnetic pole having a second base end contacting a secondmagnet, and a second tip end smaller than said second base end, therebyconcentrating magnetic flux generated by said second magnet to saidsecond tip end; wherein said first and second tip ends being apart andacross from each other to form magnetic field in between, whereas afirst surface of said first tip end and a second surface of said secondtip end converging into a convex; a channel contacting said first andsecond surfaces by a third surface that conformally encompasses saidconvex; and wherein said biological entities with magnetic labels areseparated towards said convex from said fluid sample passing throughsaid channel.
 2. The device according to claim 1, wherein said firstbase end contacts north pole surface of said first magnet and saidsecond base end contacts south pole surface of said second magnet. 3.The device according to claim 2, wherein a soft magnetic shield contactssouth pole surface of said first magnet and north pole surface of saidsecond magnet, thereby producing a magnetic flux path within said softmagnetic shield between said first and second magnets.
 4. The deviceaccording to claim 1, wherein said first magnet is the same magnet asthe said second magnet, wherein said first base end contacts north polesurface of said same magnet and said second base end contacts south polesurface of said same magnet.
 5. The device according to claim 1, whereinsaid third surface of said channel maintains being conformal to saidconvex when said channel is separated from said convex.
 6. The deviceaccording to claim 5, wherein said channel has a fourth surface oppositeto said third surface around said channel, wherein wall of said channelhas a first thickness at said fourth surface and a second thickness atsaid third surface, wherein said first thickness is larger than saidsecond thickness.
 7. The device according to claim 1, wherein saidchannel is a flexible channel whereas said third surface becomesnon-conformal to said convex when said channel is separated from saidconvex.
 8. The device according to claim 7, wherein a holder attaches toa fourth surface of said channel by a holder bottom surface, whereinsaid fourth surface being opposite to said third surface around saidchannel.
 9. The device according to claim 8, wherein said holder forcessaid channel against said convex to cause said third surface of saidchannel to become conformal to said convex.
 10. The device according toclaim 7, wherein said holder bottom surface is substantially conformalto said convex.
 11. The device according to claim 7, wherein a spacingbetween said holder bottom surface and said convex is any one of:between 0 mm to 1 mm; between 1 mm to 3 mm; between two times of thewall thickness of said channel to three times of the wall thickness ofthe said channel; and between three times of the wall thickness of saidchannel to five times of the wall thickness of said channel.
 12. Thedevice according to claim 1, wherein said first and second magnets arepermanent magnets composed of at least one of: Fe, Co, Ni, Ir, Mn, Nd,B, Sm, and Al.
 13. The device according to claim 1, wherein saidbiological entities are bound with fluorescent labels.
 14. The deviceaccording to claim 1, wherein said biological entities are any of:cells, bacteria, particles, DNA, RNA, and molecules.
 15. A method toseparate biological entities with magnetic labels from a fluid samplecomprising steps of: passing said fluid sample through a channelpositioned in contact with a separation magnet by a first surface ofsaid channel; said separation magnet separating said biological entitieswith magnetic labels from said fluid sample to form a magneticconglomerate on internal surface of said channel opposing said firstsurface; detaching said channel from said separation magnet andpositioning said channel in contact with a second magnet by a secondsurface of said channel; and said second magnet causing dissociation ofsaid magnetic conglomerate.
 16. The method according to claim 15,wherein said separation magnet comprises: a first soft magnetic polecontacting a first magnet and concentrating magnetic flux from saidfirst magnet to a first tip end of said first soft magnetic pole; asecond soft magnetic pole contacting a second magnet and concentratingmagnetic flux from said second magnet to a second tip end of said secondsoft magnetic pole; and wherein said first and second tip ends beingapart and across from each other, with a third surface of said first tipend and a fourth surface of said second tip end converging into aconvex; and wherein said first surface of said channel contacting saidthird and fourth surface and conformally encompassing said convex. 17.The method according to claim 15, wherein said second surface isopposite to said first surface around said channel
 18. A method toseparate biological entities with magnetic labels from a fluid samplecomprising steps of: passing said fluid sample through a channelpositioned in contact with a separation magnet by a first surface ofsaid channel; said separation magnet separating said biological entitieswith magnetic labels from said fluid sample to form a magneticconglomerate on internal surface of said channel opposing said firstsurface; detaching said channel from said separation magnet andpositioning said channel in contact with a mechanical excitation means;and said mechanical excitation means producing turbulence in said fluidsample contained in said channel and causing dissociation of saidmagnetic conglomerate.
 19. The method according to claim 18, whereinsaid mechanical excitation means includes one of: a motor producingvibration; a piezoelectric element producing ultrasound vibration; and amechanical structure producing stretch, compression or twisting of saidchannel.
 20. The method according to claim 18, wherein said channel isattached to a channel holder and said mechanical excitation means is incontact with said channel holder.